Antibodies binding to CD3 and CD19

ABSTRACT

The present invention generally relates to antibodies that bind to CD3 and CD19, e.g. for activating T cells. In addition, the present invention relates to polynucleotides encoding such antibodies, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the antibodies, and to methods of using them in the treatment of disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit to European Patent Application No.20181056.1, filed Jun. 19, 2020, the entire contents of which isincorporated herein by reference.

SEQUENCE LISTING

This application contains a Sequence Listing, which has been submittedelectronically via EFS-Web in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 9, 2021, isnamed P36171-US_sequence_listing.txt and is 85,241 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to antibodies that bind to CD3and CD19, e.g. for activating T cells. In addition, the presentinvention relates to polynucleotides encoding such antibodies, andvectors and host cells comprising such polynucleotides. The inventionfurther relates to methods for producing the antibodies, and to methodsof using them in the treatment of disease.

BACKGROUND

CD3 (cluster of differentiation 3) is a protein complex composed of foursubunits, the CD3γ chain, the CD3δ chain, and two CD3ε chains. CD3associates with the T-cell receptor and the ζ chain to generate anactivation signal in T lymphocytes.

CD3 has been extensively explored as drug target. Monoclonal antibodiestargeting CD3 have been used as immunosuppressant therapies inautoimmune diseases such as type I diabetes, or in the treatment oftransplant rejection. The CD3 antibody muromonab-CD3 (OKT3) was thefirst monoclonal antibody ever approved for clinical use in humans, in1985.

A more recent application of CD3 antibodies is in the form of bispecificantibodies, binding CD3 on the one hand and a target cell antigen suchas CD19 on the other hand. The simultaneous binding of such an antibodyto both of its targets will force a temporary interaction between targetcell and T cell, causing activation of any cytotoxic T cell andsubsequent lysis of the target cell. Bispecific antibodies binding toCD3 and CD19 are described e.g. in WO 2017/055314

For therapeutic purposes, an important requirement that antibodies haveto fulfill is sufficient stability both in vitro (for storage of thedrug) an in vivo (after administration to the patient). Modificationslike asparagine deamidation are typical degradations for recombinantantibodies and can affect both in vitro stability and in vivo biologicalfunctions.

Given the tremendous therapeutic potential of antibodies, particularlybispecific antibodies for the activation of T cells, there is a need forCD3 antibodies, including multispecific antibodies, with optimizedproperties.

SUMMARY OF THE INVENTION

The present invention provides antibodies, including multispecific (e.g.bispecific) antibodies, that bind to CD3 and are resistant todegradation by e.g. asparagine deamidation and thus particularly stableas required for therapeutic purposes. The (multispecific) antibodiesprovided further combine good efficacy and producibility with lowtoxicity and favorable pharmacokinetic properties.

As is shown herein, the antibodies, including multispecific antibodies,that bind to CD3, provided by the present invention, retain more thanabout 90% binding activity to CD3 after 2 weeks at pH 7.4, 37° C.,relative to the binding activity after 2 weeks at pH 6, −80° C., asdetermined by surface plasmon resonance (SPR).

In one aspect, the invention provides an antibody that binds to CD3 andCD19, wherein the antibody comprises (a) a first antigen binding domainthat binds to CD3, comprising a heavy chain variable region (VH)comprising a heavy chain complementary determining region (HCDR) 1 ofSEQ ID NO: 2, a HCDR 2 of SEQ ID NO: 3, and a HCDR 3 of SEQ ID NO: 5,and a light chain variable region (VL) comprising a light chaincomplementarity determining region (LCDR) 1 of SEQ ID NO: 8, a LCDR 2 ofSEQ ID NO: 9 and a LCDR 3 of SEQ ID NO: 10; and (b) a second andoptionally a third antigen binding domain that binds to CD19. In oneaspect, the VH of the first antigen binding domain comprises an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 7, and/or the VL ofthe first antigen binding domain comprises an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 11.

In a further aspect, the invention provides an antibody that binds toCD3 and CD19, wherein the antibody comprises (a) a first antigen bindingdomain that binds to CD3 comprising a VH sequence of SEQ ID NO: 7 and aVL sequence of SEQ ID NO: 11; and (b) a second and optionally a thirdantigen binding domain that binds to CD19.

In one aspect, the first, the second and/or, where present, the thirdantigen binding domain is a Fab molecule.

In one aspect, the antibody comprises an Fc domain composed of a firstand a second subunit.

In one aspect the first antigen binding domain is a Fab molecule whereinthe variable domains VL and VH or the constant domains CL and CH1,particularly the variable domains VL and VH, of the Fab light chain andthe Fab heavy chain are replaced by each other.

In one aspect the second and, where present, the third antigen bindingdomain is a conventional Fab molecule.

In one aspect, the second and, where present, the third antigen bindingdomain is a Fab molecule wherein in the constant domain CL the aminoacid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat) and theamino acid at position 123 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat), and in theconstant domain CH1 the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In one aspect, the first and the second antigen binding domain are fusedto each other, optionally via a peptide linker.

In one aspect, the first and the second antigen binding domain are eacha Fab molecule and either (i) the second antigen binding domain is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the Fabheavy chain of the first antigen binding domain, or (ii) the firstantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingdomain.

In one aspect, the first, the second and, where present, the thirdantigen binding domain are each a Fab molecule and the antibodycomprises an Fc domain composed of a first and a second subunit; andwherein either (i) the second antigen binding domain is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding domain and the first antigen bindingdomain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, or (ii) the firstantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingdomain and the second antigen binding domain is fused at the C-terminusof the Fab heavy chain to the N-terminus of the first subunit of the Fcdomain; and the third antigen binding domain, where present, is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the secondsubunit of the Fc domain.

In one aspect, the Fc domain is an IgG, particularly an IgG₁, Fc domain.In one aspect the Fc domain is a human Fc domain. In one aspect, the Fccomprises a modification promoting the association of the first and thesecond subunit of the Fc domain. In one aspect, the Fc domain comprisesone or more amino acid substitution that reduces binding to an Fcreceptor and/or effector function.

In one aspect, the second and, where present, the third antigen bindingdomain comprises (i) a VH comprising a HCDR 1 of SEQ ID NO: 15, a HCDR 2of SEQ ID NO: 16, and a HCDR 3 of SEQ ID NO: 17, and a VL comprising aLCDR 1 of SEQ ID NO: 19, a LCDR 2 of SEQ ID NO: 20 and a LCDR 3 of SEQID NO: 21; or (ii) a VH comprising a HCDR 1 of SEQ ID NO: 28, a HCDR 2of SEQ ID NO: 29, and a HCDR 3 of SEQ ID NO: 30, and a VL comprising aLCDR 1 of SEQ ID NO: 32, a LCDR 2 of SEQ ID NO: 33 and a LCDR 3 of SEQID NO: 34. In one aspect, the second and, where present, the thirdantigen binding domain comprises (i) a VH comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 18, and/or a VLcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22;or (ii) a VH comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 31, and/or a VL comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 35.

According to a further aspect of the invention there is provided anisolated polynucleotide encoding an antibody of the invention, and ahost cell comprising the isolated polynucleotide of the invention.

In another aspect is provided a method of producing an antibody thatbinds to CD3 and CD19, comprising the steps of (a) culturing the hostcell of the invention under conditions suitable for the expression ofthe antibody and optionally (b) recovering the antibody. The inventionalso encompasses an antibody that binds to CD3 and CD19 produced by themethod of the invention. The invention further provides a pharmaceuticalcomposition comprising the antibody of the invention and apharmaceutically acceptable carrier.

Also encompassed by the invention are methods of using the antibody andpharmaceutical composition of the invention. In one aspect the inventionprovides an antibody or pharmaceutical composition according to theinvention for use as a medicament. In one aspect is provided an antibodyor pharmaceutical composition according to the invention for use in thetreatment of a disease. Also provided is the use of an antibody orpharmaceutical composition according to the invention in the manufactureof a medicament, and the use of an antibody or pharmaceuticalcomposition according to the invention in the manufacture of amedicament for the treatment of a disease. The invention also provides amethod of treating a disease in an individual, comprising administeringto said individual an effective amount of the antibody or pharmaceuticalcomposition according to the invention. In certain aspects the diseaseis cancer. In other aspects the disease is an autoimmune disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Exemplary configurations of the (multispecific) antibodies ofthe invention. (A, D) Illustration of the “1+1 CrossMab” molecule. (B,E) Illustration of the “2+1 IgG Crossfab” molecule with alternativeorder of Crossfab and Fab components (“inverted”). (C, F) Illustrationof the “2+1 IgG Crossfab” molecule. (G, K) Illustration of the “1+1 IgGCrossfab” molecule with alternative order of Crossfab and Fab components(“inverted”). (H, L) Illustration of the “1+1 IgG Crossfab” molecule.(I, M) Illustration of the “2+1 IgG Crossfab” molecule with twoCrossFabs. (J, N) Illustration of the “2+1 IgG Crossfab” molecule withtwo CrossFabs and alternative order of Crossfab and Fab components(“inverted”). (O, S) Illustration of the “Fab-Crossfab” molecule. (P, T)Illustration of the “Crossfab-Fab” molecule. (Q, U) Illustration of the“(Fab)₂-Crossfab” molecule. (R, V) Illustration of the “Crossfab-(Fab)₂”molecule. (W, Y) Illustration of the “Fab-(Crossfab)₂” molecule. (X, Z)Illustration of the “(Crossfab)₂-Fab” molecule. Black dot: optionalmodification in the Fc domain promoting heterodimerization. ++, −−:amino acids of opposite charges optionally introduced in the CH1 and CLdomains. Crossfab molecules are depicted as comprising an exchange of VHand VL regions, but may—in aspects wherein no charge modifications areintroduced in CH1 and CL domains—alternatively comprise an exchange ofthe CH1 and CL domains.

FIG. 2. Relative binding activity of original and optimized CD3 binders,CD3_(orig) and CD3_(opt), to recombinant CD3 as measured by SPR inunstressed condition, after 14 d at 40° C. pH 6, or after 14 d at 37° C.pH 7.4 (IgG format).

FIG. 3. Binding of original and optimized CD3 binders, CD3_(orig) andCD3_(opt), to Jurkat NFAT cells as measured by flow cytometry (IgGformat). Antibodies bound to Jurkat NFAT cells were detected with afluorescently labeled anti-human Fc specific secondary antibody.

FIG. 4. Schematic illustration of the CD3 activation assay used inExample 3.

FIG. 5. Jurkat NFAT activation with original and optimized CD3 binders,CD3_(orig) and CD3_(opt) (IgG format). Jurkat NFAT reporter cells wereco-incubated with anti-PGLALA expressing CHO (CHO-PGLALA) cells in thepresence of CD3_(orig) or CD3_(opt) IgG PGLALA, or CD3_(opt) IgG wt asnegative control. CD3 activation was quantified by measuringluminescence after 24 h.

FIG. 6. (A) Schematic illustration of the T-cell bispecific antibody(TCB) molecules used in the Examples. All tested TCB antibody moleculeswere produced as “2+1 IgG CrossFab, inverted” with charge modifications(VH/VL exchange in CD3 binder, charge modifications in target antigenbinders, EE=147E, 213E; RK=123R, 124K). (B-E) Components for theassembly of the TCB: light chain of anti-TYRP1 Fab molecule with chargemodifications in CH1 and CL (B), light chain of anti-CD3 crossover Fabmolecule (C), heavy chain with knob and PG LALA mutations in Fc region(D), heavy chain with hole and PG LALA mutations in Fc region (E).

FIG. 7. Binding of CD19-TCB antibodies to CD3-expressing Jurkat cells(A) and to CD19-expressing Z-138 (B) and Nalm-6 (C) cells, as measuredby flow cytometry.

FIG. 8. Target-specific killing of CD19+ target cells induced byCD19-TCB antibodies. (A) Z-138 target cells, (B) Nalm-6 target cells.

FIG. 9. T cell activation induced by CD19-TCB antibodies after killingof Z-138 target cells. (A) CD25 expression on CD4 T cells, (B) CD69expression on CD4 T cells, (C) CD107 expression on CD4 T cells, (D) CD25expression on CD8 T cells, (E) CD69 expression on CD8 T cells, (F) CD107expression on CD8 T cells.

FIG. 10. T cell activation induced by CD19-TCB antibodies after killingof Nalm-6 target cells. (A) CD25 expression on CD4 T cells, (B) CD69expression on CD4 T cells, (C) CD107 expression on CD4 T cells, (D) CD25expression on CD8 T cells, (E) CD69 expression on CD8 T cells, (F) CD107expression on CD8 T cells.

FIG. 11. Design of the in vivo study of Example 10.

FIG. 12. Body weight change upon treatment with different doses ofCD19-TCB or CD20-TCB, in the study of Example 10. n=3 mice per group.Mean+/−SEM.

FIG. 13. Cytokine release in serum at 4 hours after treatment withCD19-TCB or CD20-TCB, with or without obinituzumab (Gazyva(R)pre-treatment (GPT), in the study of Example 10. (A) MIP-1β, (B) IL-6,(C) IFN-γ, (D) IL-5, (E) GM-CSF, (F) TNF-α, (G) IL-2, (H) IL-1β, (I)IL-13, (J) MCP1, (K) IL-8, (L) IL-10, (M) G-CSF, (N) IL-12p70, (O)IL-17. Bars in each panel from left to right: CD19-TCB 0.5 mg/kg,CD19-TCB 0.15 mg/kg, CD19-TCB 0.05 mg/kg, GPT+CD19-TCB 0.5 mg/kg,CD20-TCB 0.15 mg/kg, GPT+CD20-TCB 0.15 mg/kg. Mean+SEM.

FIG. 14. B cell counts in blood at 4 hours, 24 hours and 72 hours aftertreatment CD19-TCB or CD20-TCB, with or without obinituzumab (Gazyva®)pre-treatment (GPT), in the study of Example 10. Mean+SEM.

FIG. 15. Treatment schedule and experimental set-up. Humanized NSG micewere subcutaneously engrafted with a lymphoma patient-derived xenograft(PDX) (5 million cells). Tumor volumes were calculated from calipermeasurements. When they reached 200 mm³, mice were randomized in groupsof 8 based on their tumor size. Mice were then weekly injected (i.v.)with vehicle or 0.5 mg/kg CD19-TCB.

FIG. 16. Effect of CD19-TCB treatment on tumor growth. Tumor volumeswere calculated from caliper measurements two (volume <1000 mm³) orthree times (volume ≥1000 mm³) per week for n=7 mice in group B and n=8mice in group A, as described in FIG. 15. Mean+SD with *p≤0.05,**p≤0.01, ***p≤0.001 by Mann-Whitney test. Arrows indicate each of the 4treatments with CD19-TCB or vehicle.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Terms are used herein as generally used in the art, unless otherwisedefined in the following. As used herein, the terms “first”, “second” or“third” with respect to antigen binding domains etc., are used forconvenience of distinguishing when there is more than one of each typeof moiety. Use of these terms is not intended to confer a specific orderor orientation of the moiety unless explicitly so stated.

The terms “anti-CD3 antibody” and “an antibody that binds to CD3” referto an antibody that is capable of binding CD3 with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting CD3. In one aspect, the extent of binding of ananti-CD3 antibody to an unrelated, non-CD3 protein is less than about10% of the binding of the antibody to CD3 as measured, e.g., by surfaceplasmon resonance (SPR). In certain aspects, an antibody that binds toCD3 has a dissociation constant (K_(D)) of ≤1 μM, ≤500 nM, ≤200 nM, or≤100 nM. An antibody is said to “specifically bind” to CD3 when theantibody has a K_(D) of 1 μM or less, as measured, e.g., by SPR. Incertain aspects, an anti-CD3 antibody binds to an epitope of CD3 that isconserved among CD3 from different species.

Similarly, the terms “anti-CD19 antibody” and “an antibody that binds toCD19” refer to an antibody that is capable of binding CD19 withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting CD19. In one aspect, the extent ofbinding of an anti-CD19 antibody to an unrelated, non-CD19 protein isless than about 10% of the binding of the antibody to CD19 as measured,e.g., by surface plasmon resonance (SPR). In certain aspects, anantibody that binds to CD19 has a dissociation constant (K_(D)) of ≤1μM, ≤500 nM, ≤200 nM, or ≤100 nM. An antibody is said to “specificallybind” to CD19 when the antibody has a K_(D) of 1 μM or less, asmeasured, e.g., by SPR. In certain aspects, an anti-CD19 antibody bindsto an epitope of CD19 that is conserved among CD19 from differentspecies.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv and scFab), single-domain antibodies, and multispecificantibodies formed from antibody fragments. For a review of certainantibody fragments, see Hollinger and Hudson, Nature Biotechnology23:1126-1136 (2005).

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprised in the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example,monoclonal antibodies may be made by a variety of techniques, includingbut not limited to the hybridoma method, recombinant DNA methods,phage-display methods, and methods utilizing transgenic animalscontaining all or part of the human immunoglobulin loci, such methodsand other exemplary methods for making monoclonal antibodies beingdescribed herein.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some aspects, an antibody is purified togreater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC, affinity chromatography, size exclusion chromatography) methods.For review of methods for assessment of antibody purity, see, e.g.,Flatman et al., J. Chromatogr. B 848:79-87 (2007). In some aspects, theantibodies provided by the present invention are isolated antibodies.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human CDRs and amino acid residues from humanFRs. In certain aspects, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDRs correspond to those of anon-human antibody, and all or substantially all of the FRs correspondto those of a human antibody. Such variable domains are referred toherein as “humanized variable region”. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. In some aspects, some FR residues in a humanizedantibody are substituted with corresponding residues from a non-humanantibody (e.g., the antibody from which the CDR residues are derived),e.g., to restore or improve antibody specificity or affinity. A“humanized form” of an antibody, e.g. of a non-human antibody, refers toan antibody that has undergone humanization.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues. In certain aspects, ahuman antibody is derived from a non-human transgenic mammal, forexample a mouse, a rat, or a rabbit. In certain aspects, a humanantibody is derived from a hybridoma cell line. Antibodies or antibodyfragments isolated from human antibody libraries are also consideredhuman antibodies or human antibody fragments herein.

The term “antigen binding domain” refers to the part of an antibody thatcomprises the area which binds to and is complementary to part or all ofan antigen. An antigen binding domain may be provided by, for example,one or more antibody variable domains (also called antibody variableregions). In preferred aspects, an antigen binding domain comprises anantibody light chain variable domain (VL) and an antibody heavy chainvariable domain (VH).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and complementarity determining regions (CDRs). See, e.g.,Kindt et al., Kuby Immunology, 6^(th) ed., W.H. Freeman & Co., page 91(2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). As usedherein in connection with variable region sequences, “Kabat numbering”refers to the numbering system set forth by Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991).

As used herein, the amino acid positions of all constant regions anddomains of the heavy and light chain are numbered according to the Kabatnumbering system described in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991), referred to as “numberingaccording to Kabat” or “Kabat numbering” herein. Specifically the Kabatnumbering system (see pages 647-660 of Kabat, et al., Sequences ofProteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) is used for thelight chain constant domain CL of kappa and lambda isotype and the KabatEU index numbering system (see pages 661-723) is used for the heavychain constant domains (CH1, hinge, CH2 and CH3), which is hereinfurther clarified by referring to “numbering according to Kabat EUindex” or “Kabat EU index numbering” in this case. The term“hypervariable region” or “HVR”, as used herein, refers to each of theregions of an antibody variable domain which are hypervariable insequence and which determine antigen binding specificity, for example“complementarity determining regions” (“CDRs”). Generally, antibodiescomprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three inthe VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include:

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat et al., Sequences of Proteins of Immunological Interest,        5th Ed. Public Health Service, National Institutes of Health,        Bethesda, Md. (1991)); and    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)).

Unless otherwise indicated, the CDRs are determined according to Kabatet al., supra. One of skill in the art will understand that the CDRdesignations can also be determined according to Chothia, supra,McCallum, supra, or any other scientifically accepted nomenclaturesystem. “Framework” or “FR” refers to variable domain residues otherthan complementarity determining regions (CDRs). The FR of a variabledomain generally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingorder in VH (or VL):FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4. Unless otherwiseindicated, CDR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some aspects, the number of amino acid changes is 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some aspects, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence. A “humanconsensus framework” is a framework which represents the most commonlyoccurring amino acid residues in a selection of human immunoglobulin VLor VH framework sequences. Generally, the selection of humanimmunoglobulin VL or VH sequences is from a subgroup of variable domainsequences. Generally, the subgroup of sequences is a subgroup as inKabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.

The term “immunoglobulin molecule” herein refers to a protein having thestructure of a naturally occurring antibody. For example,immunoglobulins of the IgG class are heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two light chains and two heavy chainsthat are disulfide-bonded. From N- to C-terminus, each heavy chain has avariable domain (VH), also called a variable heavy domain or a heavychain variable region, followed by three constant domains (CH1, CH2, andCH3), also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable domain (VL), also called avariable light domain or a light chain variable region, followed by aconstant light (CL) domain, also called a light chain constant region.The heavy chain of an immunoglobulin may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of animmunoglobulin may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Animmunoglobulin essentially consists of two Fab molecules and an Fcdomain, linked via the immunoglobulin hinge region.

The “class” of an antibody or immunoglobulin refers to the type ofconstant domain or constant region possessed by its heavy chain. Thereare five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively.

A “Fab molecule” refers to a protein consisting of the VH and CH1 domainof the heavy chain (the “Fab heavy chain”) and the VL and CL domain ofthe light chain (the “Fab light chain”) of an immunoglobulin.

By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fabmolecule wherein the variable domains or the constant domains of the Fabheavy and light chain are exchanged (i.e. replaced by each other), i.e.the crossover Fab molecule comprises a peptide chain composed of thelight chain variable domain VL and the heavy chain constant domain 1 CH1(VL-CH1, in N- to C-terminal direction), and a peptide chain composed ofthe heavy chain variable domain VH and the light chain constant domainCL (VH-CL, in N- to C-terminal direction). For clarity, in a crossoverFab molecule wherein the variable domains of the Fab light chain and theFab heavy chain are exchanged, the peptide chain comprising the heavychain constant domain 1 CH1 is referred to herein as the “heavy chain”of the (crossover) Fab molecule. Conversely, in a crossover Fab moleculewherein the constant domains of the Fab light chain and the Fab heavychain are exchanged, the peptide chain comprising the heavy chainvariable domain VH is referred to herein as the “heavy chain” of the(crossover) Fab molecule.

In contrast thereto, by a “conventional” Fab molecule is meant a Fabmolecule in its natural format, i.e. comprising a heavy chain composedof the heavy chain variable and constant domains (VH-CH1, in N- toC-terminal direction), and a light chain composed of the light chainvariable and constant domains (VL-CL, in N- to C-terminal direction).

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. In one aspect, a human IgGheavy chain Fc region extends from Cys226, or from Pro230, to thecarboxyl-terminus of the heavy chain. However, antibodies produced byhost cells may undergo post-translational cleavage of one or more,particularly one or two, amino acids from the C-terminus of the heavychain. Therefore, an antibody produced by a host cell by expression of aspecific nucleic acid molecule encoding a full-length heavy chain mayinclude the full-length heavy chain, or it may include a cleaved variantof the full-length heavy chain. This may be the case where the final twoC-terminal amino acids of the heavy chain are glycine (G446) and lysine(K447, numbering according to Kabat EU index). Therefore, the C-terminallysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447),of the Fc region may or may not be present. Amino acid sequences ofheavy chains including an Fc region (or a subunit of an Fc domain asdefined herein) are denoted herein without C-terminal glycine-lysinedipeptide if not indicated otherwise. In one aspect, a heavy chainincluding an Fc region (subunit) as specified herein, comprised in anantibody according to the invention, comprises an additional C-terminalglycine-lysine dipeptide (G446 and K447, numbering according to Kabat EUindex). In one aspect, a heavy chain including an Fc region (subunit) asspecified herein, comprised in an antibody according to the invention,comprises an additional C-terminal glycine residue (G446, numberingaccording to Kabat EU index). Unless otherwise specified herein,numbering of amino acid residues in the Fc region or constant region isaccording to the EU numbering system, also called the EU index, asdescribed in Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991 (see also above). A “subunit” of an Fc domain asused herein refers to one of the two polypeptides forming the dimeric Fcdomain, i.e. a polypeptide comprising C-terminal constant regions of animmunoglobulin heavy chain, capable of stable self-association. Forexample, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgGCH3 constant domain. By “fused” is meant that the components (e.g. a Fabmolecule and an Fc domain subunit) are linked by peptide bonds, eitherdirectly or via one or more peptide linkers.

The term “multispecific” means that the antibody is able to specificallybind to at least two distinct antigenic determinants. A multispecificantibody can be, for example, a bispecific antibody. Typically, abispecific antibody comprises two antigen binding sites, each of whichis specific for a different antigenic determinant. In certain aspectsthe multispecific (e.g. bispecific) antibody is capable ofsimultaneously binding two antigenic determinants, particularly twoantigenic determinants expressed on two distinct cells.

The term “valent” as used herein denotes the presence of a specifiednumber of antigen binding sites in an antigen binding molecule. As such,the term “monovalent binding to an antigen” denotes the presence of one(and not more than one) antigen binding site specific for the antigen inthe antigen binding molecule.

An “antigen binding site” refers to the site, i.e. one or more aminoacid residues, of an antigen binding molecule which provides interactionwith the antigen. For example, the antigen binding site of an antibodycomprises amino acid residues from the complementarity determiningregions (CDRs). A native immunoglobulin molecule typically has twoantigen binding sites, a Fab molecule typically has a single antigenbinding site.

As used herein, the term “antigenic determinant” or “antigen” refers toa site (e.g. a contiguous stretch of amino acids or a conformationalconfiguration made up of different regions of non-contiguous aminoacids) on a polypeptide macromolecule to which an antigen binding domainbinds, forming an antigen binding domain-antigen complex. Usefulantigenic determinants can be found, for example, on the surfaces oftumor cells, on the surfaces of virus-infected cells, on the surfaces ofother diseased cells, on the surface of immune cells, free in bloodserum, and/or in the extracellular matrix (ECM). In a preferred aspect,the antigen is a human protein.

“CD3” refers to any native CD3 from any vertebrate source, includingmammals such as primates (e.g. humans), non-human primates (e.g.cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwiseindicated. The term encompasses “full-length,” unprocessed CD3 as wellas any form of CD3 that results from processing in the cell. The termalso encompasses naturally occurring variants of CD3, e.g., splicevariants or allelic variants. In one aspect, CD3 is human CD3,particularly the epsilon subunit of human CD3 (CD3c). The amino acidsequence of human CD3c is shown in SEQ ID NO: 45 (without signalpeptide). See also UniProt (www.uniprot.org) accession no. P07766(version 209), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_000724.1. Inanother aspect, CD3 is cynomolgus (Macaca fascicularis) CD3,particularly cynomolgus CD3E. The amino acid sequence of cynomolgus CD3εis shown in SEQ ID NO: 46 (without signal peptide). See also NCBIGenBank no. BAB71849.1. In certain aspects the antibody of the inventionbinds to an epitope of CD3 that is conserved among the CD3 antigens fromdifferent species, particularly human and cynomolgus CD3. In preferredaspects, the antibody binds to human CD3.

A “target cell antigen” as used herein refers to an antigenicdeterminant presented on the surface of a target cell, for example aB-cell. Preferably, the target cell antigen is not CD3, and/or isexpressed on a different cell than CD3. According to the invention, thetarget cell antigen is CD19, particularly human CD19.

“CD19” stands for cluster of differentiation 19 (also known asB-lymphocyte antigen CD19 or B-lymphocyte surface antigen B4) and refersto any native CD19 from any vertebrate source, including mammals such asprimates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) androdents (e.g. mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed CD19 as well as any form of CD19that results from processing in the cell. The term also encompassesnaturally occurring variants of CD19, e.g., splice variants or allelicvariants. In one aspect, CD19 is human CD19. See for the human proteinUniProt (www.uniprot.org) accession no. P15391 (version 211), or NCBI(www.ncbi.nlm.nih.gov/) RefSeq NP_001761.3. In certain aspects theantibody of the invention binds to an epitope of CD19 that is conservedamong the CD19 antigens from different species, particularly human andcynomolgus CD19. In preferred aspects, the antibody binds to human CD19.

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., an antibody and an antigen). The affinity of amolecule X for its partner Y can generally be represented by thedissociation constant (K_(D)). Affinity can be measured bywell-established methods known in the art, including those describedherein. A preferred method for measuring affinity is Surface PlasmonResonance (SPR).

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more complementary determining regions (CDRs),compared to a parent antibody which does not possess such alterations,such alterations resulting in an improvement in the affinity of theantibody for antigen.

“Reduced binding”, for example reduced binding to an Fc receptor, refersto a decrease in affinity for the respective interaction, as measuredfor example by SPR. For clarity, the term includes also reduction of theaffinity to zero (or below the detection limit of the analytic method),i.e. complete abolishment of the interaction. Conversely, “increasedbinding” refers to an increase in binding affinity for the respectiveinteraction.

“T cell activation” as used herein refers to one or more cellularresponse of a T lymphocyte, particularly a cytotoxic T lymphocyte,selected from: proliferation, differentiation, cytokine secretion,cytotoxic effector molecule release, cytotoxic activity, and expressionof activation markers. Suitable assays to measure T cell activation areknown in the art and described herein. A “modification promoting theassociation of the first and the second subunit of the Fc domain” is amanipulation of the peptide backbone or the post-translationalmodifications of an Fc domain subunit that reduces or prevents theassociation of a polypeptide comprising the Fc domain subunit with anidentical polypeptide to form a homodimer. A modification promotingassociation as used herein preferably includes separate modificationsmade to each of the two Fc domain subunits desired to associate (i.e.the first and the second subunit of the Fc domain), wherein themodifications are complementary to each other so as to promoteassociation of the two Fc domain subunits. For example, a modificationpromoting association may alter the structure or charge of one or bothof the Fc domain subunits so as to make their association sterically orelectrostatically favorable, respectively. Thus, (hetero)dimerizationoccurs between a polypeptide comprising the first Fc domain subunit anda polypeptide comprising the second Fc domain subunit, which may benon-identical in the sense that further components fused to each of thesubunits (e.g. antigen binding domains) are not the same. In someaspects, the modification promoting the association of the first and thesecond subunit of the Fc domain comprises an amino acid mutation in theFc domain, specifically an amino acid substitution. In a preferredaspect, the modification promoting the association of the first and thesecond subunit of the Fc domain comprises a separate amino acidmutation, specifically an amino acid substitution, in each of the twosubunits of the Fc domain. The term “effector functions” refers to thosebiological activities attributable to the Fc region of an antibody,which vary with the antibody isotype. Examples of antibody effectorfunctions include: C1q binding and complement dependent cytotoxicity(CDC), Fc receptor binding, antibody-dependent cell-mediatedcytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP),cytokine secretion, immune complex-mediated antigen uptake by antigenpresenting cells, down regulation of cell surface receptors (e.g. B cellreceptor), and B cell activation.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc domain of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Humanactivating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa(CD32), and FcαRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immunemechanism leading to the lysis of antibody-coated target cells by immuneeffector cells. The target cells are cells to which antibodies orderivatives thereof comprising an Fc region specifically bind, generallyvia the protein part that is N-terminal to the Fc region. As usedherein, the term “reduced ADCC” is defined as either a reduction in thenumber of target cells that are lysed in a given time, at a givenconcentration of antibody in the medium surrounding the target cells, bythe mechanism of ADCC defined above, and/or an increase in theconcentration of antibody in the medium surrounding the target cells,required to achieve the lysis of a given number of target cells in agiven time, by the mechanism of ADCC. The reduction in ADCC is relativeto the ADCC mediated by the same antibody produced by the same type ofhost cells, using the same standard production, purification,formulation and storage methods (which are known to those skilled in theart), but that has not been engineered. For example, the reduction inADCC mediated by an antibody comprising in its Fc domain an amino acidsubstitution that reduces ADCC, is relative to the ADCC mediated by thesame antibody without this amino acid substitution in the Fc domain.Suitable assays to measure ADCC are well known in the art (see e.g. PCTpublication no. WO 2006/082515 or PCT publication no. WO 2012/130831).

As used herein, the terms “engineer, engineered, engineering”, areconsidered to include any manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpolypeptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches.

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., reduced bindingto an Fc receptor, or increased association with another peptide. Aminoacid sequence deletions and insertions include amino- and/orcarboxy-terminal deletions and insertions of amino acids. Preferredamino acid mutations are amino acid substitutions. For the purpose ofaltering e.g. the binding characteristics of an Fc region,non-conservative amino acid substitutions, i.e. replacing one amino acidwith another amino acid having different structural and/or chemicalproperties, are particularly preferred. Amino acid substitutions includereplacement by non-naturally occurring amino acids or by naturallyoccurring amino acid derivatives of the twenty standard amino acids(e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine,5-hydroxylysine). Amino acid mutations can be generated using genetic orchemical methods well known in the art. Genetic methods may includesite-directed mutagenesis, PCR, gene synthesis and the like. It iscontemplated that methods of altering the side chain group of an aminoacid by methods other than genetic engineering, such as chemicalmodification, may also be useful. Various designations may be usedherein to indicate the same amino acid mutation. For example, asubstitution from proline at position 329 of the Fc domain to glycinecan be indicated as 329G, G329, G₃₂₉, P329G, or Pro329Gly.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR)software or the FASTA program package. Those skilled in the art candetermine appropriate parameters for aligning sequences, including anyalgorithms needed to achieve maximal alignment over the full length ofthe sequences being compared. Alternatively, the percent identity valuescan be generated using the sequence comparison computer program ALIGN-2.The ALIGN-2 sequence comparison computer program was authored byGenentech, Inc., and the source code has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087and is described in WO 2001/007611.

Unless otherwise indicated, for purposes herein, % amino acid sequenceidentity values are generated using the ggsearch program of the FASTApackage version 36.3.8c or later with a BLOSUM50 comparison matrix. TheFASTA program package was authored by W. R. Pearson and D. J. Lipman(“Improved Tools for Biological Sequence Analysis”, PNAS 85 (1988)2444-2448), W. R. Pearson (“Effective protein sequence comparison” Meth.Enzymol. 266 (1996) 227-258), and Pearson et. al. (Genomics 46 (1997)24-36) and is publicly available fromwww.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml orwww.ebi.ac.uk/Tools/sss/fasta. Alternatively, a public server accessibleat fasta.bioch.virginia.edu/fast_awww2/index.cgi can be used to comparethe sequences, using the ggsearch (global protein:protein) program anddefault options (BLOSUM50; open: −10; ext: −2; Ktup=2) to ensure aglobal, rather than local, alignment is performed. Percent amino acididentity is given in the output alignment header.

The term “polynucleotide” or “nucleic acid molecule” includes anycompound and/or substance that comprises a polymer of nucleotides. Eachnucleotide is composed of a base, specifically a purine- or pyrimidinebase (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil(U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group.Often, the nucleic acid molecule is described by the sequence of bases,whereby said bases represent the primary structure (linear structure) ofa nucleic acid molecule. The sequence of bases is typically representedfrom 5′ to 3′. Herein, the term nucleic acid molecule encompassesdeoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) andgenomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA),synthetic forms of DNA or RNA, and mixed polymers comprising two or moreof these molecules. The nucleic acid molecule may be linear or circular.In addition, the term nucleic acid molecule includes both, sense andantisense strands, as well as single stranded and double stranded forms.Moreover, the herein described nucleic acid molecule can containnaturally occurring or non-naturally occurring nucleotides. Examples ofnon-naturally occurring nucleotides include modified nucleotide baseswith derivatized sugars or phosphate backbone linkages or chemicallymodified residues. Nucleic acid molecules also encompass DNA and RNAmolecules which are suitable as a vector for direct expression of anantibody of the invention in vitro and/or in vivo, e.g., in a host orpatient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can beunmodified or modified. For example, mRNA can be chemically modified toenhance the stability of the RNA vector and/or expression of the encodedmolecule so that mRNA can be injected into a subject to generate theantibody in vivo (see e.g., Stadler et al. (2017) Nature Medicine23:815-817, or EP 2 101 823 B1).

An “isolated” nucleic acid molecule refers to a nucleic acid moleculethat has been separated from a component of its natural environment. Anisolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that ordinarily contain the nucleic acid molecule,but the nucleic acid molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation.

“Isolated polynucleotide (or nucleic acid) encoding an antibody” refersto one or more polynucleotide molecules encoding antibody heavy andlight chains (or fragments thereof), including such polynucleotidemolecule(s) in a single vector or separate vectors, and suchpolynucleotide molecule(s) present at one or more locations in a hostcell.

The term “vector”, as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe antibodies of the present invention. Host cells include culturedcells, e.g. mammalian cultured cells, such as HEK cells, CHO cells, BHKcells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myelomacells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insectcells, and plant cells, to name only a few, but also cells comprisedwithin a transgenic animal, transgenic plant or cultured plant or animaltissue. In one aspect, the host cell of the invention is a eukaryoticcell, particularly a mammalian cell. In one aspect, the host cell is nota cell within a human body.

The term “pharmaceutical composition” or “pharmaceutical formulation”refers to a preparation which is in such form as to permit thebiological activity of an active ingredient contained therein to beeffective, and which contains no additional components which areunacceptably toxic to a subject to which the composition would beadministered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition or formulation, other than an activeingredient, which is nontoxic to a subject. A pharmaceuticallyacceptable carrier includes, but is not limited to, a buffer, excipient,stabilizer, or preservative. As used herein, “treatment” (andgrammatical variations thereof such as “treat” or “treating”) refers toclinical intervention in an attempt to alter the natural course of adisease in the individual being treated, and can be performed either forprophylaxis or during the course of clinical pathology. Desirableeffects of treatment include, but are not limited to, preventingoccurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. In some aspects, antibodies of theinvention are used to delay development of a disease or to slow theprogression of a disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). In certain aspects, theindividual or subject is a human.

An “effective amount” of an agent, e.g., a pharmaceutical composition,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

II. Compositions and Methods

The invention provides antibodies that bind CD3 and CD19. The antibodiesshow superior stability, combined with other favorable properties fortherapeutic application, e.g. with respect to efficacy and safety,pharmacokinetics, as well as producibility. Antibodies of the inventionas useful, e.g., for the treatment of diseases such as cancer orautoimmune disease.

A. Anti-CD3/CD19 Antibodies

In one aspect, the invention provides antibodies that bind to CD3 andCD19. In one aspect, provided are isolated antibodies that bind to CD3and CD19. In one aspect, the invention provides antibodies thatspecifically bind to CD3 and CD19. In certain aspects, the anti-CD3/CD19antibodies retain more than about 90% binding activity to CD3 after 2weeks at pH 7.4, 37° C., relative to the binding activity after 2 weeksat pH 6, −80° C., as determined by surface plasmon resonance (SPR).

In one aspect, the invention provides an antibody that binds to CD3 andCD19, wherein the antibody comprises (a) a first antigen binding domainthat binds to CD3, comprising a heavy chain variable region (VH)comprising a heavy chain complementary determining region (HCDR) 1 ofSEQ ID NO: 2, a HCDR 2 of SEQ ID NO: 3, and a HCDR 3 of SEQ ID NO: 5,and a light chain variable region (VL) comprising a light chaincomplementarity determining region (LCDR) 1 of SEQ ID NO: 8, a LCDR 2 ofSEQ ID NO: 9 and a LCDR 3 of SEQ ID NO: 10.

In one aspect, the antibody is a humanized antibody. In one aspect, thefirst antigen binding domain is a humanized antigen binding domain (i.e.an antigen binding domain of a humanized antibody).

In one aspect, the VH and/or the VL of the first antigen binding domainis a humanized variable region.

In one aspect, the VH and/or the VL of the first antigen binding domaincomprises an acceptor human framework, e.g. a human immunoglobulinframework or a human consensus framework.

In one aspect, the VH of the first antigen binding domain comprises oneor more heavy chain framework sequence (i.e. the FR1, FR2, FR3 and/orFR4 sequence) of the heavy chain variable region sequence of SEQ ID NO:7. In one aspect, the VH of the first antigen binding domain comprisesan amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of SEQ ID NO: 7. In one aspect, theVH of the first antigen binding domain comprises an amino acid sequencethat is at least about 95% identical to the amino acid sequence of SEQID NO: 7. In one aspect, the VH of the first antigen binding domaincomprises an amino acid sequence that is at least about 98% identical tothe amino acid sequence of SEQ ID NO: 7. In certain aspects, a VHsequence having at least 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an antibody comprisingthat sequence retains the ability to bind to CD3. In certain aspects, atotal of 1 to 10 amino acids have been substituted, inserted and/ordeleted in the amino acid sequence of SEQ ID NO: 7. In certain aspects,substitutions, insertions, or deletions occur in regions outside theCDRs (i.e., in the FRs). In one aspect, the VH of the first antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 7.Optionally, the VH of the first antigen binding domain comprises theamino acid sequence of SEQ ID NO: 7, including post-translationalmodifications of that sequence.

In one aspect, the VL of the first antigen binding domain comprises oneor more light chain framework sequence (i.e. the FR1, FR2, FR3 and/orFR4 sequence) of the light chain variable region sequence of SEQ ID NO:11. In one aspect, the VL of the first antigen binding domain comprisesan amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of SEQ ID NO: 11. In one aspect,the VL of the first antigen binding domain comprises an amino acidsequence that is at least about 95% identical to the amino acid sequenceof SEQ ID NO: 11. In one aspect, the VL of the first antigen bindingdomain comprises an amino acid sequence that is at least about 98%identical to the amino acid sequence of SEQ ID NO: 11. In certainaspects, a VL sequence having at least 95%, 96%, 97%, 98%, or 99%identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but anantibody comprising that sequence retains the ability to bind to CD3. Incertain aspects, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in the amino acid sequence of SEQ ID NO: 11. Incertain aspects, substitutions, insertions, or deletions occur inregions outside the CDRs (i.e., in the FRs). In one aspect, the VL ofthe first antigen binding domain comprises the amino acid sequence ofSEQ ID NO: 11. Optionally, the VL of the first antigen binding domaincomprises the amino acid sequence of SEQ ID NO: 11, includingpost-translational modifications of that sequence.

In one aspect, the VH of the first antigen binding domain comprises anamino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of SEQ ID NO: 7, and the VL of thefirst antigen binding domain comprises an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence of SEQ ID NO: 11. In one aspect, the VH of the first antigenbinding domain comprises the amino acid sequence of SEQ ID NO: 7 and theVL of the first antigen binding domain comprises the amino acid sequenceof SEQ ID NO: 11.

In a further aspect, the invention provides an antibody that binds toCD3 and CD19, wherein the antibody comprises a first antigen bindingdomain that binds to CD3 comprising a VH comprising the amino acidsequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence ofSEQ ID NO: 11.

In a further aspect, the invention provides an antibody that binds toCD3 and CD19, wherein the antibody comprises a first antigen bindingdomain that binds to CD3 comprising a VH sequence of SEQ ID NO: 7 and aVL sequence of SEQ ID NO: 11.

In another aspect, the invention provides an antibody that binds to CD3and CD19, wherein the antibody comprises a first antigen binding domainthat binds to CD3 comprising a VH comprising the heavy chain CDRsequences of the VH of SEQ ID NO: 7, and a VL comprising the light chainCDR sequences of the VL of SEQ ID NO: 11.

In a further aspect, the first antigen binding domain comprises theHCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 7and the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ IDNO: 11.

In one aspect, the VH of the first antigen binding domain comprises theheavy chain CDR sequences of the VH of SEQ ID NO: 7 and a framework ofat least 95%, 96%, 97%, 98% or 99% sequence identity to the frameworksequence of the VH of SEQ ID NO: 7. In one aspect, the VH of the firstantigen binding domain comprises the heavy chain CDR sequences of the VHof SEQ ID NO: 7 and a framework of at least 95% sequence identity to theframework sequence of the VH of SEQ ID NO: 7. In another aspect, the VHof the first antigen binding domain comprises the heavy chain CDRsequences of the VH of SEQ ID NO: 7 and a framework of at least 98%sequence identity to the framework sequence of the VH of SEQ ID NO: 7.

In one aspect, the VL of the first antigen binding domain comprises thelight chain CDR sequences of the VL of SEQ ID NO: 11 and a framework ofat least 95%, 96%, 97%, 98% or 99% sequence identity to the frameworksequence of the VL of SEQ ID NO: 11. In one aspect, the VL of the firstantigen binding domain comprises the light chain CDR sequences of the VLof SEQ ID NO: 11 and a framework of at least 95% sequence identity tothe framework sequence of the VL of SEQ ID NO: 11. In another aspect,the VL of the first antigen binding domain comprises the light chain CDRsequences of the VL of SEQ ID NO: 11 and a framework of at least 98%sequence identity to the framework sequence of the VL of SEQ ID NO: 11.

In one aspect, the invention provides an antibody that binds to CD3 andCD19, wherein the antibody comprises a first antigen binding domain thatbinds to CD3 comprising a VH sequence as in any of the aspects providedabove, and a VL sequence as in any of the aspects provided above. In oneaspect, the antibody comprises a human constant region. In one aspect,the antibody is an immunoglobulin molecule comprising a human constantregion, particularly an IgG class immunoglobulin molecule comprising ahuman CH1, CH2, CH3 and/or CL domain. Exemplary sequences of humanconstant domains are given in SEQ ID NOs 52 and 53 (human kappa andlambda CL domains, respectively) and SEQ ID NO: 54 (human IgG₁ heavychain constant domains CH1-CH2-CH3). In one aspect, the antibodycomprises a light chain constant region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 53,particularly the amino acid sequence of SEQ ID NO: 52. In one aspect,the antibody comprises a heavy chain constant region comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 54. Particularly, theheavy chain constant region may comprise amino acid mutations in the Fcdomain as described herein.

In one aspect, the first antigen binding domain comprises a humanconstant region. In one aspect, the first antigen binding moiety is aFab molecule comprising a human constant region, particularly a humanCH1 and/or CL domain. In one aspect, the first antigen binding domaincomprises a light chain constant region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 53,particularly the amino acid sequence of SEQ ID NO: 52. Particularly, thelight chain constant region may comprise amino acid mutations asdescribed herein under “charge modifications” and/or may comprisedeletion or substitutions of one or more (particularly two)N-terminalamino acids if in a crossover Fab molecule. In some aspects, the firstantigen binding domain comprises a heavy chain constant regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the CH1 domain sequence comprised in theamino acid sequence of SEQ ID NO: 54. Particularly, the heavy chainconstant region (specifically CH1 domain) may comprise amino acidmutations as described herein under “charge modifications”.

In one aspect, the antibody is a monoclonal antibody.

In one aspect, the antibody is an IgG, particularly an IgG₁, antibody.In one aspect, the antibody is a full-length antibody.

In another aspect, the antibody is an antibody fragment selected fromthe group of an Fv molecule, a scFv molecule, a Fab molecule, and aF(ab′)₂ molecule; particularly a Fab molecule. In another aspect, theantibody fragment is a diabody, a triabody or a tetrabody.

In one aspect, the first antigen binding domain is a Fab molecule. In apreferred aspect the first antigen binding domain is a Fab moleculewherein the variable domains VL and VH or the constant domains CL andCH1, particularly the variable domains VL and VH, of the Fab light chainand the Fab heavy chain are replaced by each other (i.e. the firstantigen binding domain is a crossover Fab molecule).

In a further aspect, the antibody according to any of the above aspectsmay incorporate any of the features, singly or in combination, asdescribed in sections II. A. 1.-8. below.

In a preferred aspect, the antibody comprises an Fc domain, particularlyan IgG Fc domain, more particularly an IgG₁ Fc domain. In one aspect theFc domain is a human Fc domain. In one aspect, the Fc domain is a humanIgG₁ Fc domain. The Fc domain is composed of a first and a secondsubunit and may incorporate any of the features, singly or incombination, described hereinbelow in relation to Fc domain variants(section II. A. 8.).

According to the invention, the antibody comprises a second andoptionally a third antigen binding domain which binds to CD19 (i.e. theantibody is a multispecific antibody, as further described hereinbelow(section II. A. 7.).

1. Antibody Fragments

In certain aspects, an antibody provided herein is an antibody fragment.

In one aspect, the antibody fragment is a Fab, Fab′, Fab′-SH, or F(ab′)₂molecule, in particular a Fab molecule as described herein. “Fab′molecule” differ from Fab molecules by the addition of residues at thecarboxy terminus of the CH1 domain including one or more cysteines fromthe antibody hinge region. Fab′-SH are Fab′ molecules in which thecysteine residue(s) of the constant domains bear a free thiol group.Pepsin treatment yields an F(ab′)₂ molecule that has two antigen-bindingsites (two Fab molecules) and a part of the Fc region.

In another aspect, the antibody fragment is a diabody, a triabody or atetrabody. “Diabodies” are antibody fragments with two antigen-bindingsites that may be bivalent or bispecific. See, for example, EP 404,097;WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollingeret al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies andtetrabodies are also described in Hudson et al., Nat. Med. 9:129-134(2003).

In a further aspect, the antibody fragment is a single chain Fabmolecule. A “single chain Fab molecule” or “scFab” is a polypeptideconsisting of an antibody heavy chain variable domain (VH), an antibodyheavy chain constant domain 1 (CH1), an antibody light chain variabledomain (VL), an antibody light chain constant domain (CL) and a linker,wherein said antibody domains and said linker have one of the followingorders in N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b)VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL.In particular, said linker is a polypeptide of at least 30 amino acids,preferably between 32 and 50 amino acids. Said single chain Fabmolecules are stabilized via the natural disulfide bond between the CLdomain and the CH1 domain. In addition, these single chain Fab moleculesmight be further stabilized by generation of interchain disulfide bondsvia insertion of cysteine residues (e.g., position 44 in the variableheavy chain and position 100 in the variable light chain according toKabat numbering).

In another aspect, the antibody fragment is single-chain variablefragment (scFv). A “single-chain variable fragment” or “scFv” is afusion protein of the variable domains of the heavy (VH) and lightchains (VL) of an antibody, connected by a linker. In particular, thelinker is a short polypeptide of 10 to 25 amino acids and is usuallyrich in glycine for flexibility, as well as serine or threonine forsolubility, and can either connect the N-terminus of the VH with theC-terminus of the VL, or vice versa. This protein retains thespecificity of the original antibody, despite removal of the constantregions and the introduction of the linker. For a review of scFvfragments, see, e.g., Plückthun, in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, NewYork), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos.5,571,894 and 5,587,458.

In another aspect, the antibody fragment is a single-domain antibody.“Single-domain antibodies” are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain aspects, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asrecombinant production by recombinant host cells (e.g., E. coli), asdescribed herein.

2. Humanized Antibodies

In certain aspects, an antibody provided herein is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which the CDRs (or portions thereof) arederived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome aspects, some FR residues in a humanized antibody are substitutedwith corresponding residues from a non-human antibody (e.g., theantibody from which the CDR residues are derived), e.g., to restore orimprove antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing specificity determining region(SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osboum et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

3. Glycosylation Variants

In certain aspects, an antibody provided herein is altered to increaseor decrease the extent to which the antibody is glycosylated. Additionor deletion of glycosylation sites to an antibody may be convenientlyaccomplished by altering the amino acid sequence such that one or moreglycosylation sites is created or removed.

Where the antibody comprises an Fc region, the oligosaccharide attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some aspects, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one aspect, antibody variants are provided having a non-fucosylatedoligosaccharide, i.e. an oligosaccharide structure that lacks fucoseattached (directly or indirectly) to an Fc region. Such non-fucosylatedoligosaccharide (also referred to as “afucosylated” oligosaccharide)particularly is an N-linked oligosaccharide which lacks a fucose residueattached to the first GlcNAc in the stem of the biantennaryoligosaccharide structure. In one aspect, antibody variants are providedhaving an increased proportion of non-fucosylated oligosaccharides inthe Fc region as compared to a native or parent antibody. For example,the proportion of non-fucosylated oligosaccharides may be at least about20%, at least about 40%, at least about 60%, at least about 80%, or evenabout 100% (i.e. no fucosylated oligosaccharides are present). Thepercentage of non-fucosylated oligosaccharides is the (average) amountof oligosaccharides lacking fucose residues, relative to the sum of alloligosaccharides attached to Asn 297 (e.g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2006/082515, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (EUnumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such antibodies having an increased proportion ofnon-fucosylated oligosaccharides in the Fc region may have improvedFcγRIIIa receptor binding and/or improved effector function, inparticular improved ADCC function. See, e.g., US 2003/0157108; US2004/0093621.

Examples of cell lines capable of producing antibodies with reducedfucosylation include Lec13 CHO cells deficient in protein fucosylation(Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US2003/0157108; and WO 2004/056312, especially at Example 11), andknockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8,knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng.87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688(2006); and WO 2003/085107), or cells with reduced or abolished activityof a GDP-fucose synthesis or transporter protein (see, e.g.,US2004259150, US2005031613, US2004132140, US2004110282).

In a further aspect, antibody variants are provided with bisectedoligosaccharides, e.g., in which a biantennary oligosaccharide attachedto the Fc region of the antibody is bisected by GlcNAc. Such antibodyvariants may have reduced fucosylation and/or improved ADCC function asdescribed above. Examples of such antibody variants are described, e.g.,in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al.,Biotechn Bioeng 93, 851-861 (2006); WO 99/54342; WO 2004/065540, WO2003/011878.

Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

4. Cysteine Engineered Antibody Variants

In certain aspects, it may be desirable to create cysteine engineeredantibodies, e.g., THIOMAB™ antibodies, in which one or more residues ofan antibody are substituted with cysteine residues. In preferredaspects, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. Cysteine engineered antibodies may begenerated as described, e.g., in U.S. Pat. Nos. 7,521,541, 8,30,930,7,855,275, 9,000,130, or WO 2016040856.

5. Antibody Derivatives

In certain aspects, an antibody provided herein may be further modifiedto contain additional non-proteinaceous moieties that are known in theart and readily available. The moieties suitable for derivatization ofthe antibody include but are not limited to water soluble polymers.Non-limiting examples of water soluble polymers include, but are notlimited to, polyethylene glycol (PEG), copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

6. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-CD3/CD19antibody herein conjugated (chemically bonded) to one or moretherapeutic agents such as cytotoxic agents, chemotherapeutic agents,drugs, growth inhibitory agents, toxins (e.g., protein toxins,enzymatically active toxins of bacterial, fungal, plant, or animalorigin, or fragments thereof), or radioactive isotopes.

In one aspect, an immunoconjugate is an antibody-drug conjugate (ADC) inwhich an antibody is conjugated to one or more of the therapeutic agentsmentioned above. The antibody is typically connected to one or more ofthe therapeutic agents using linkers. An overview of ADC technologyincluding examples of therapeutic agents and drugs and linkers is setforth in Pharmacol Review 68:3-19 (2016).

In another aspect, an immunoconjugate comprises an antibody of theinvention conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another aspect, an immunoconjugate comprises an antibody of theinvention conjugated to a radioactive atom to form a radioconjugate. Avariety of radioactive isotopes are available for the production ofradioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸,Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu. When theradioconjugate is used for detection, it may comprise a radioactive atomfor scintigraphic studies, for example Tc^(99m) or I¹²³ or a spin labelfor nuclear magnetic resonance (NMR) imaging (also known as magneticresonance imaging, MRI), such as I¹²³, I¹³¹, In¹¹¹, F¹⁹, C¹³, N¹⁵, O¹⁷,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO 94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

7. Multispecific Antibodies

An antibody provided herein is a multispecific antibody, particularly abispecific antibody. Multispecific antibodies are monoclonal antibodiesthat have binding specificities for at least two different antigenicdeterminants (e.g., two different proteins, or two different epitopes onthe same protein). In certain aspects, the multispecific antibody hasthree or more binding specificities. According to the present invention,one of the binding specificities is for CD3 and the other specificity isfor CD19.

Multispecific antibodies may be prepared as full length antibodies orantibody fragments. Techniques for making multispecific antibodiesinclude, but are not limited to, recombinant co-expression of twoimmunoglobulin heavy chain-light chain pairs having differentspecificities (see Milstein and Cuello, Nature 305: 537 (1983)) and“knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168, andAtwell et al., J. Mol. Biol. 270:26 (1997)). Multi-specific antibodiesmay also be made by engineering electrostatic steering effects formaking antibody Fc-heterodimeric molecules (see, e.g., WO 2009/089004);cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat.No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); usingleucine zippers to produce bi-specific antibodies (see, e.g., Kostelnyet al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011/034605); usingthe common light chain technology for circumventing the light chainmis-pairing problem (see, e.g., WO 98/50431); using “diabody” technologyfor making bispecific antibody fragments (see, e.g., Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chainFv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368(1994)); and preparing trispecific antibodies as described, e.g., inTutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more antigen binding sites,including for example, “Octopus antibodies”, or DVD-Ig are also includedherein (see, e.g., WO 2001/77342 and WO 2008/024715). Other examples ofmultispecific antibodies with three or more antigen binding sites can befound in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792,and WO 2013/026831. The multispecific antibody or antigen bindingfragment thereof also includes a “Dual Acting FAb” or “DAF” comprisingan antigen binding site that binds to CD3 as well as another differentantigen, or two different epitopes of CD3 (see, e.g., US 2008/0069820and WO 2015/095539).

Multi-specific antibodies may also be provided in an asymmetric formwith a domain crossover in one or more binding arms of the same antigenspecificity (so-called “CrossMab” technology), i.e. by exchanging theVH/VL domains (see e.g., WO 2009/080252 and WO 2015/150447), the CH1/CLdomains (see e.g., WO 2009/080253) or the complete Fab arms (see e.g.,WO 2009/080251, WO 2016/016299, also see Schaefer et al, PNAS, 108(2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). AsymmetricalFab arms can also be engineered by introducing charged or non-chargedamino acid mutations into domain interfaces to direct correct Fabpairing. See e.g., WO 2016/172485.

Various further molecular formats for multispecific antibodies are knownin the art and are included herein (see e.g., Spiess et al., Mol Immunol67 (2015) 95-106).

A particular type of multispecific antibodies are bispecific antibodiesdesigned to simultaneously bind to a surface antigen on a target cell,e.g., a B cell, and to an activating, invariant component of the T cellreceptor (TCR) complex, such as CD3, for retargeting of T cells to killtarget cells. Hence, the antibody provided herein is a multispecificantibody, particularly a bispecific antibody, wherein one of the bindingspecificities is for CD3 and the other is for CD19 as the target cellantigen.

Examples of bispecific antibody formats that may be useful for thispurpose include, but are not limited to, the so-called “BiTE”(bispecific T cell engager) molecules wherein two scFv molecules arefused by a flexible linker (see, e.g., WO 2004/106381, WO 2005/061547,WO 2007/042261, and WO 2008/119567, Nagorsen and Bäuerle, Exp Cell Res317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305(1996)) and derivatives thereof, such as tandem diabodies (“TandAb”;Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); “DART” (dual affinityretargeting) molecules which are based on the diabody format but featurea C-terminal disulfide bridge for additional stabilization (Johnson etal., J Mol Biol 399, 436-449 (2010)), and so-called triomabs, which arewhole hybrid mouse/rat IgG molecules (reviewed in Seimetz et al., CancerTreat Rev 36, 458-467 (2010)). Particular T cell bispecific antibodyformats included herein are described in WO 2013/026833, WO 2013/026839,WO 2016/020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

Preferred aspects of the antibody of the present invention are describedin the following.

In one aspect, the invention provides an antibody that binds to CD3 andCD19, comprising a first antigen binding domain that binds to CD3, asdescribed herein, and comprising a second and optionally a third antigenbinding domain that binds to CD19.

According to preferred aspects of the invention, the antigen bindingdomains comprised in the antibody are Fab molecules (i.e. antigenbinding domains composed of a heavy and a light chain, each comprising avariable and a constant domain). In one aspect, the first, the secondand/or, where present, the third antigen binding domain is a Fabmolecule. In one aspect, said Fab molecule is human. In a preferredaspect, said Fab molecule is humanized. In yet another aspect, said Fabmolecule comprises human heavy and light chain constant domains.

Preferably, at least one of the antigen binding domains is a crossoverFab molecule. Such modification reduces mispairing of heavy and lightchains from different Fab molecules, thereby improving the yield andpurity of the (multispecific) antibody of the invention in recombinantproduction. In a preferred crossover Fab molecule useful for the(multispecific) antibody of the invention, the variable domains of theFab light chain and the Fab heavy chain (VL and VH, respectively) areexchanged. Even with this domain exchange, however, the preparation ofthe (multispecific) antibody may comprise certain side products due to aso-called Bence Jones-type interaction between mispaired heavy and lightchains (see Schaefer et al, PNAS, 108 (2011) 11187-11191). To furtherreduce mispairing of heavy and light chains from different Fab moleculesand thus increase the purity and yield of the desired (multispecific)antibody, charged amino acids with opposite charges may be introduced atspecific amino acid positions in the CH1 and CL domains of either theFab molecule binding to CD3, or the Fab molecule(s) binding to CD19, asfurther described herein. Charge modifications are made either in theconventional Fab molecule(s) comprised in the (multispecific) antibody(such as shown e.g. in FIGS. 1 A-C, G-J), or in the VH/VL crossover Fabmolecule(s) comprised in the (multispecific) antibody (such as showne.g. in FIG. 1 D-F, K-N) (but not in both). In preferred aspects, thecharge modifications are made in the conventional Fab molecule(s)comprised in the (multispecific) antibody (which in preferred aspectsbind(s) to CD19).

In a preferred aspect according to the invention, the (multispecific)antibody is capable of simultaneous binding to CD3 and CD19. In oneaspect, the (multispecific) antibody is capable of crosslinking a T celland a target cell by simultaneous binding to CD3 and CD19. In an evenmore preferred aspect, such simultaneous binding results in lysis of thetarget cell, particularly a CD19-expressing target cell such as aB-cell. In one aspect, such simultaneous binding results in activationof the T cell. In other aspects, such simultaneous binding results in acellular response of a T lymphocyte, particularly a cytotoxic Tlymphocyte, selected from the group of: proliferation, differentiation,cytokine secretion, cytotoxic effector molecule release, cytotoxicactivity, and expression of activation markers. In one aspect, bindingof the (multispecific) antibody to CD3 without simultaneous binding toCD19 does not result in T cell activation.

In one aspect, the (multispecific) antibody is capable of re-directingcytotoxic activity of a T cell to a target cell. In a preferred aspect,said re-direction is independent of MHC-mediated peptide antigenpresentation by the target cell and and/or specificity of the T cell.

Preferably, a T cell according to any of the aspects of the invention isa cytotoxic T cell. In some aspects the T cell is a CD4+ or a CD8+ Tcell, particularly a CD8+ T cell.

a) First Antigen Binding Domain

The (multispecific) antibody of the invention comprises at least oneantigen binding domain (the first antigen binding domain) that binds toCD3. In preferred aspects, CD3 is human CD3 (SEQ ID NO: 45) orcynomolgus CD3 (SEQ ID NO: 46) most particularly human CD3. In oneaspect the first antigen binding domain is cross-reactive for (i.e.specifically binds to) human and cynomolgus CD3. In some aspects, CD3 isthe epsilon subunit of CD3 (CD3 epsilon).

In a preferred aspect, the (multispecific) antibody comprises not morethan one antigen binding domain that binds to CD3. In one aspect the(multispecific) antibody provides monovalent binding to CD3.

In one aspect, the antigen binding domain that binds to CD3 is anantibody fragment selected from the group of an Fv molecule, a scFvmolecule, a Fab molecule, and a F(ab′)₂ molecule. In a preferred aspect,the antigen binding domain that binds to CD3 is a Fab molecule.

In preferred aspects, the antigen binding domain that binds to CD3 is acrossover Fab molecule as described herein, i.e. a Fab molecule whereinthe variable domains VH and VL or the constant domains CH1 and CL of theFab heavy and light chains are exchanged/replaced by each other. In suchaspects, the antigen binding domain(s) that binds to CD19 is preferablya conventional Fab molecule. In aspects where there is more than oneantigen binding domain, particularly Fab molecule, that binds to CD19comprised in the (multispecific) antibody, the antigen binding domainthat binds to CD3 preferably is a crossover Fab molecule and the antigenbinding domain that bind to CD19 are conventional Fab molecules.

In alternative aspects, the antigen binding domain that binds to CD3 isa conventional Fab molecule. In such aspects, the antigen bindingdomain(s) that binds CD19 is a crossover Fab molecule as describedherein, i.e. a Fab molecule wherein the variable domains VH and VL orthe constant domains CH1 and CL of the Fab heavy and light chains areexchanged/replaced by each other. In aspects where there is more thanone antigen binding domain, particularly Fab molecule, that binds to CD3comprised in the (multispecific) antibody, the antigen binding domainthat binds to CD19 preferably is a crossover Fab molecule and theantigen binding domains that bind to CD3 are conventional Fab molecules.

In preferred aspects, the first antigen binding domain is a Fab moleculewherein the variable domains VL and VH or the constant domains CL andCH1, particularly the variable domains VL and VH, of the Fab light chainand the Fab heavy chain are replaced by each other (i.e. according tosuch aspect, the first antigen binding domain is a crossover Fabmolecule wherein the variable or constant domains of the Fab light chainand the Fab heavy chain are exchanged). In one such aspect, the second(and the third, if any) antigen binding domain is a conventional Fabmolecule. In one aspect, not more than one antigen binding domain thatbinds to CD3 is present in the (multispecific) antibody (i.e. theantibody provides monovalent binding to CD3).

b) Second (and Third) Antigen Binding Domain

The (multispecific) antibody of the invention comprises at least oneantigen binding domain (the second and optionally the third antigenbinding domain), particularly a Fab molecule, that binds to CD19. Thesecond antigen binding domain is able to direct the (multispecific)antibody to a target site, for example to a specific type of cell thatexpresses CD19.

In one aspect, the antigen binding domain that binds to CD19 is anantibody fragment selected from the group of an Fv molecule, a scFvmolecule, a Fab molecule, and a F(ab′)₂ molecule. In a preferred aspect,the antigen binding domain that binds to CD19 is a Fab molecule.

In certain aspects, the (multispecific) antibody comprises two antigenbinding domains, particularly Fab molecules, that bind to CD19. In apreferred aspect, all of these antigen binding domains are identical,i.e. they have the same molecular format (e.g. conventional or crossoverFab molecule) and comprise the same amino acid sequences including thesame amino acid substitutions in the CH1 and CL domain as describedherein (if any). In one aspect, the (multispecific) antibody comprisesnot more than two antigen binding domains, particularly Fab molecules,that bind to CD19.

In preferred aspects, the antigen binding domain(s) that bind to CD19is/are a conventional Fab molecule. In such aspects, the antigen bindingdomain(s) that binds to CD3 is a crossover Fab molecule as describedherein, i.e. a Fab molecule wherein the variable domains VH and VL orthe constant domains CH1 and CL of the Fab heavy and light chains areexchanged/replaced by each other.

In alternative aspects, the antigen binding domain(s) that bind to CD19is/are a crossover Fab molecule as described herein, i.e. a Fab moleculewherein the variable domains VH and VL or the constant domains CH1 andCL of the Fab heavy and light chains are exchanged/replaced by eachother. In such aspects, the antigen binding domain(s) that binds to CD3is a conventional Fab molecule.

In one aspect, the second (and, where present, third) antigen bindingdomain comprises a human constant region. In one aspect, the second(and, where present, third) antigen binding domain is a Fab moleculecomprising a human constant region, particularly a human CH1 and/or CLdomain. Exemplary sequences of human constant domains are given in SEQID NOs 52 and 53 (human kappa and lambda CL domains, respectively) andSEQ ID NO: 54 (human IgG₁ heavy chain constant domains CH1-CH2-CH3). Inone aspect, the second (and, where present, third) antigen bindingdomain comprises a light chain constant region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 53,particularly the amino acid sequence of SEQ ID NO: 52. Particularly, thelight chain constant region may comprise amino acid mutations asdescribed herein under “charge modifications” and/or may comprisedeletion or substitutions of one or more (particularly two)N-terminalamino acids if in a crossover Fab molecule. In some aspects, the second(and, where present, third) antigen binding domain comprises a heavychain constant region comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the CH1 domainsequence comprised in the amino acid sequence of SEQ ID NO: 54.Particularly, the heavy chain constant region (specifically CH1 domain)may comprise amino acid mutations as described herein under “chargemodifications”.

In one aspect, the second (and, where present, the third) antigenbinding domain comprises a heavy chain variable region (VH) comprising aheavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 15,a HCDR 2 of SEQ ID NO: 16, and a HCDR 3 of SEQ ID NO: 17, and a lightchain variable region (VL) comprising a light chain complementaritydetermining region (LCDR) 1 of SEQ ID NO: 19, a LCDR 2 of SEQ ID NO: 20and a LCDR 3 of SEQ ID NO: 21.

In one aspect, the second (and, where present, third) antigen bindingdomain is (derived from) a humanized antibody. In one aspect, the second(and, where present, third) antigen binding domain is a humanizedantigen binding domain (i.e. an antigen binding domain of a humanizedantibody). In one aspect, the VH and/or the VL of the second (and, wherepresent, third) antigen binding domain is a humanized variable region.

In one aspect, the VH and/or the VL of the second (and, where present,third) antigen binding domain comprises an acceptor human framework,e.g. a human immunoglobulin framework or a human consensus framework.

In one aspect, the VH of the second (and, where present, the third)antigen binding domain comprises one or more heavy chain frameworksequence (i.e. the FR1, FR2, FR3 and/or FR4 sequence) of SEQ ID NO: 18.In one aspect, the VH of the second (and, where present, the third)antigen binding domain comprises an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO: 18. In one aspect, the VH of the second (and, where present,the third) antigen binding domain comprises an amino acid sequence thatis at least about 95% identical to the amino acid sequence of SEQ ID NO:18. In one aspect, the VH of the second (and, where present, the third)antigen binding domain comprises an amino acid sequence that is at leastabout 98% identical to the amino acid sequence of SEQ ID NO: 18. Incertain aspects, a VH sequence having at least 95%, 96%, 97%, 98%, or99% identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but anantibody comprising that sequence retains the ability to bind to CD19.In certain aspects, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in the amino acid sequence of SEQID NO: 18. In certain aspects, substitutions, insertions, or deletionsoccur in regions outside the CDRs (i.e., in the FRs). In one aspect, theVH of the second (and, where present, the third) antigen binding domaincomprises the amino acid sequence of SEQ ID NO: 18. Optionally, the VHof the second (and, where present, the third) antigen binding domaincomprises the amino acid sequence of SEQ ID NO: 18, includingpost-translational modifications of that sequence.

In one aspect, the VL of the second (and, where present, the third)antigen binding domain comprises one or more light chain frameworksequence (i.e. the FR1, FR2, FR3 and/or FR4 sequence) of SEQ ID NO: 22.In one aspect, the VL of the second (and, where present, the third)antigen binding domain comprises an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO: 22. In one aspect, the VL of the second (and, where present,the third) antigen binding domain comprises an amino acid sequence thatis at least about 95% identical to the amino acid sequence of SEQ ID NO:22. In one aspect, the VL of the second (and, where present, the third)antigen binding domain comprises an amino acid sequence that is at leastabout 98% identical to the amino acid sequence of SEQ ID NO: 22. Incertain aspects, a VL sequence having at least 95%, 96%, 97%, 98%, or99% identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but anantibody comprising that sequence retains the ability to bind to CD19.In certain aspects, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in the amino acid sequence of SEQID NO: 22. In certain aspects, substitutions, insertions, or deletionsoccur in regions outside the CDRs (i.e., in the FRs). In one aspect, theVL of the second (and, where present, the third) antigen binding domaincomprises the amino acid sequence of SEQ ID NO: 22. Optionally, the VLof the second (and, where present, the third) antigen binding domaincomprises the amino acid sequence of SEQ ID NO: 22, includingpost-translational modifications of that sequence.

In one aspect, the VH of the second (and, where present, the third)antigen binding domain comprises an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO: 18, and the VL of the second (and, where present, the third)antigen binding domain comprises an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO: 22. In one aspect, the VH of the second (and, where present,the third) antigen binding domain comprises the amino acid sequence ofSEQ ID NO: 18 and the VL of the second (and, where present, the third)antigen binding domain comprises the amino acid sequence of SEQ ID NO:22.

In a further aspect, the second (and, where present, the third) antigenbinding domain comprises a VH comprising the sequence of SEQ ID NO: 18and a VL comprising the sequence of SEQ ID NO: 22.

In a further aspect, the second (and, where present, the third) antigenbinding domain comprises a VH sequence of SEQ ID NO: 18 and a VLsequence of SEQ ID NO: 22.

In another aspect, the second (and, where present, the third) antigenbinding domain comprises a VH comprising the heavy chain CDR sequencesof the VH of SEQ ID NO: 18, and a VL comprising the light chain CDRsequences of the VL of SEQ ID NO: 22.

In a further aspect, the second (and, where present, the third) antigenbinding domain comprises the HCDR1, HCDR2 and HCDR3 amino acid sequencesof the VH of SEQ ID NO: 18 and the LCDR1, LCDR2 and LCDR3 amino acidsequences of the VL of SEQ ID NO: 22.

In one aspect, the VH of the second (and, where present, the third)antigen binding domain comprises the heavy chain CDR sequences of the VHof SEQ ID NO: 18 and a framework of at least 95%, 96%, 97%, 98% or 99%sequence identity to the framework sequence of the VH of SEQ ID NO: 18.In one aspect, the VH of the second (and, where present, the third)antigen binding domain comprises the heavy chain CDR sequences of the VHof SEQ ID NO: 18 and a framework of at least 95% sequence identity tothe framework sequence of the VH of SEQ ID NO: 18. In another aspect,the VH of the second (and, where present, the third) antigen bindingdomain comprises the heavy chain CDR sequences of the VH of SEQ ID NO:18 and a framework of at least 98% sequence identity to the frameworksequence of the VH of SEQ ID NO: 18.

In one aspect, the VL of the second (and, where present, the third)antigen binding domain comprises the light chain CDR sequences of the VLof SEQ ID NO: 22 and a framework of at least 95%, 96%, 97%, 98% or 99%sequence identity to the framework sequence of the VL of SEQ ID NO: 22.In one aspect, the VL of the second (and, where present, the third)antigen binding domain comprises the light chain CDR sequences of the VLof SEQ ID NO: 22 and a framework of at least 95% sequence identity tothe framework sequence of the VL of SEQ ID NO: 22. In another aspect,the VL of the second (and, where present, the third) antigen bindingdomain comprises the light chain CDR sequences of the VL of SEQ ID NO:22 and a framework of at least 98% sequence identity to the frameworksequence of the VL of SEQ ID NO: 22.

In another aspect, the second (and, where present, the third) antigenbinding domain comprises a heavy chain variable region (VH) comprising aheavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 28,a HCDR 2 of SEQ ID NO: 29, and a HCDR 3 of SEQ ID NO: 30, and a lightchain variable region (VL) comprising a light chain complementaritydetermining region (LCDR) 1 of SEQ ID NO: 32, a LCDR 2 of SEQ ID NO: 33and a LCDR 3 of SEQ ID NO: 34.

In one aspect, the second (and, where present, third) antigen bindingdomain is (derived from) a humanized antibody. In one aspect, the second(and, where present, third) antigen binding domain is a humanizedantigen binding domain (i.e. an antigen binding domain of a humanizedantibody). In one aspect, the VH and/or the VL of the second (and, wherepresent, third) antigen binding domain is a humanized variable region.

In one aspect, the VH and/or the VL of the second (and, where present,third) antigen binding domain comprises an acceptor human framework,e.g. a human immunoglobulin framework or a human consensus framework.

In one aspect, the VH of the second (and, where present, the third)antigen binding domain comprises one or more heavy chain frameworksequence (i.e. the FR1, FR2, FR3 and/or FR4 sequence) of SEQ ID NO: 31.In one aspect, the VH of the second (and, where present, the third)antigen binding domain comprises an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO: 31. In one aspect, the VH of the second (and, where present,the third) antigen binding domain comprises an amino acid sequence thatis at least about 95% identical to the amino acid sequence of SEQ ID NO:31. In one aspect, the VH of the second (and, where present, the third)antigen binding domain comprises an amino acid sequence that is at leastabout 98% identical to the amino acid sequence of SEQ ID NO: 31. Incertain aspects, a VH sequence having at least 95%, 96%, 97%, 98%, or99% identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but anantibody comprising that sequence retains the ability to bind to CD19.In certain aspects, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in the amino acid sequence of SEQID NO: 31. In certain aspects, substitutions, insertions, or deletionsoccur in regions outside the CDRs (i.e., in the FRs). In one aspect, theVH of the second (and, where present, the third) antigen binding domaincomprises the amino acid sequence of SEQ ID NO: 31. Optionally, the VHof the second (and, where present, the third) antigen binding domaincomprises the amino acid sequence of SEQ ID NO: 31, includingpost-translational modifications of that sequence.

In one aspect, the VL of the second (and, where present, the third)antigen binding domain comprises one or more light chain frameworksequence (i.e. the FR1, FR2, FR3 and/or FR4 sequence) of SEQ ID NO: 35.In one aspect, the VL of the second (and, where present, the third)antigen binding domain comprises an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO: 35. In one aspect, the VL of the second (and, where present,the third) antigen binding domain comprises an amino acid sequence thatis at least about 95% identical to the amino acid sequence of SEQ ID NO:35. In one aspect, the VL of the second (and, where present, the third)antigen binding domain comprises an amino acid sequence that is at leastabout 98% identical to the amino acid sequence of SEQ ID NO: 35. Incertain aspects, a VL sequence having at least 95%, 96%, 97%, 98%, or99% identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but anantibody comprising that sequence retains the ability to bind to CD19.In certain aspects, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in the amino acid sequence of SEQID NO: 35. In certain aspects, substitutions, insertions, or deletionsoccur in regions outside the CDRs (i.e., in the FRs). In one aspect, theVL of the second (and, where present, the third) antigen binding domaincomprises the amino acid sequence of SEQ ID NO: 35. Optionally, the VLof the second (and, where present, the third) antigen binding domaincomprises the amino acid sequence of SEQ ID NO: 35, includingpost-translational modifications of that sequence.

In one aspect, the VH of the second (and, where present, the third)antigen binding domain comprises an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO: 31, and the VL of the second (and, where present, the third)antigen binding domain comprises an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO: 35. In one aspect, the VH of the second (and, where present,the third) antigen binding domain comprises the amino acid sequence ofSEQ ID NO: 31 and the VL of the second (and, where present, the third)antigen binding domain comprises the amino acid sequence of SEQ ID NO:35.

In a further aspect, the second (and, where present, the third) antigenbinding domain comprises a VH comprising the sequence of SEQ ID NO: 31and a VL comprising the sequence of SEQ ID NO: 35.

In a further aspect, the second (and, where present, the third) antigenbinding domain comprises a VH sequence of SEQ ID NO: 31 and a VLsequence of SEQ ID NO: 35.

In another aspect, the second (and, where present, the third) antigenbinding domain comprises a VH comprising the heavy chain CDR sequencesof the VH of SEQ ID NO: 31, and a VL comprising the light chain CDRsequences of the VL of SEQ ID NO: 35.

In a further aspect, the second (and, where present, the third) antigenbinding domain comprises the HCDR1, HCDR2 and HCDR3 amino acid sequencesof the VH of SEQ ID NO: 31 and the LCDR1, LCDR2 and LCDR3 amino acidsequences of the VL of SEQ ID NO: 35.

In one aspect, the VH of the second (and, where present, the third)antigen binding domain comprises the heavy chain CDR sequences of the VHof SEQ ID NO: 31 and a framework of at least 95%, 96%, 97%, 98% or 99%sequence identity to the framework sequence of the VH of SEQ ID NO: 31.In one aspect, the VH of the second (and, where present, the third)antigen binding domain comprises the heavy chain CDR sequences of the VHof SEQ ID NO: 31 and a framework of at least 95% sequence identity tothe framework sequence of the VH of SEQ ID NO: 31. In another aspect,the VH of the second (and, where present, the third) antigen bindingdomain comprises the heavy chain CDR sequences of the VH of SEQ ID NO:31 and a framework of at least 98% sequence identity to the frameworksequence of the VH of SEQ ID NO: 31.

In one aspect, the VL of the second (and, where present, the third)antigen binding domain comprises the light chain CDR sequences of the VLof SEQ ID NO: 35 and a framework of at least 95%, 96%, 97%, 98% or 99%sequence identity to the framework sequence of the VL of SEQ ID NO: 35.In one aspect, the VL of the second (and, where present, the third)antigen binding domain comprises the light chain CDR sequences of the VLof SEQ ID NO: 35 and a framework of at least 95% sequence identity tothe framework sequence of the VL of SEQ ID NO: 35. In another aspect,the VL of the second (and, where present, the third) antigen bindingdomain comprises the light chain CDR sequences of the VL of SEQ ID NO:35 and a framework of at least 98% sequence identity to the frameworksequence of the VL of SEQ ID NO: 35.

c) Charge Modifications

The (multispecific) antibody of the invention may comprise amino acidsubstitutions in Fab molecules comprised therein which are particularlyefficient in reducing mispairing of light chains with non-matching heavychains (Bence-Jones-type side products), which can occur in theproduction of Fab-based multispecific antibodies with a VH/VL exchangein one (or more, in case of molecules comprising more than twoantigen-binding Fab molecules) of their binding arms (see also PCTpublication no. WO 2015/150447, particularly the examples therein,incorporated herein by reference in its entirety). The ratio of adesired (multispecific) antibody compared to undesired side products, inparticular Bence Jones-type side products occurring in multispecificantibodies with a VH/VL domain exchange in one of their binding arms,can be improved by the introduction of charged amino acids with oppositecharges at specific amino acid positions in the CH1 and CL domains(sometimes referred to herein as “charge modifications”).

Accordingly, in some aspects wherein the first and the second (and,where present, the third) antigen binding domain of the (multispecific)antibody are both Fab molecules, and in one of the antigen bindingdomains (particularly the first antigen binding domain) the variabledomains VL and VH of the Fab light chain and the Fab heavy chain arereplaced by each other,

i) in the constant domain CL of the second (and, where present, thethird) antigen binding domain the amino acid at position 124 issubstituted by a positively charged amino acid (numbering according toKabat), and wherein in the constant domain CH1 of the second (and, wherepresent, the third) antigen binding domain the amino acid at position147 or the amino acid at position 213 is substituted by a negativelycharged amino acid (numbering according to Kabat EU index); orii) in the constant domain CL of the first antigen binding domain theamino acid at position 124 is substituted by a positively charged aminoacid (numbering according to Kabat), and wherein in the constant domainCH1 of the first antigen binding domain the amino acid at position 147or the amino acid at position 213 is substituted by a negatively chargedamino acid (numbering according to Kabat EU index).

The (multispecific) antibody does not comprise both modificationsmentioned under i) and ii). The constant domains CL and CH1 of theantigen binding domain having the VH/VL exchange are not replaced byeach other (i.e. remain unexchanged).

In a more specific aspect,

i) in the constant domain CL of the second (and, where present, thethird) antigen binding domain the amino acid at position 124 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat), and in the constant domain CH1 of thesecond (and, where present, the third) antigen binding domain the aminoacid at position 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index); orii) in the constant domain CL of the first antigen binding domain theamino acid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat), and in theconstant domain CH1 of the first antigen binding domain the amino acidat position 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In one such aspect, in the constant domain CL of the second (and, wherepresent, the third) antigen binding domain the amino acid at position124 is substituted independently by lysine (K), arginine (R) orhistidine (H) (numbering according to Kabat), and in the constant domainCH1 of the second (and, where present, the third) antigen binding domainthe amino acid at position 147 or the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In a further aspect, in the constant domain CL of the second (and, wherepresent, the third) antigen binding domain the amino acid at position124 is substituted independently by lysine (K), arginine (R) orhistidine (H) (numbering according to Kabat), and in the constant domainCH1 of the second (and, where present, the third) antigen binding domainthe amino acid at position 147 is substituted independently by glutamicacid (E), or aspartic acid (D) (numbering according to Kabat EU index).In a preferred aspect, in the constant domain CL of the second (and,where present, the third) antigen binding domain the amino acid atposition 124 is substituted independently by lysine (K), arginine (R) orhistidine (H) (numbering according to Kabat) and the amino acid atposition 123 is substituted independently by lysine (K), arginine (R) orhistidine (H) (numbering according to Kabat), and in the constant domainCH1 of the second (and, where present, the third) antigen binding domainthe amino acid at position 147 is substituted independently by glutamicacid (E), or aspartic acid (D) (numbering according to Kabat EU index)and the amino acid at position 213 is substituted independently byglutamic acid (E), or aspartic acid (D) (numbering according to Kabat EUindex).

In a more preferred aspect, in the constant domain CL of the second(and, where present, the third) antigen binding domain the amino acid atposition 124 is substituted by lysine (K) (numbering according to Kabat)and the amino acid at position 123 is substituted by lysine (K)(numbering according to Kabat), and in the constant domain CH1 of thesecond (and, where present, the third) antigen binding domain the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex).

In an even more preferred aspect, in the constant domain CL of thesecond (and, where present, the third) antigen binding domain the aminoacid at position 124 is substituted by lysine (K) (numbering accordingto Kabat) and the amino acid at position 123 is substituted by arginine(R) (numbering according to Kabat), and in the constant domain CH1 ofthe second (and, where present, the third) antigen binding domain theamino acid at position 147 is substituted by glutamic acid (E)(numbering according to Kabat EU index) and the amino acid at position213 is substituted by glutamic acid (E) (numbering according to Kabat EUindex).

In preferred aspects, if amino acid substitutions according to the aboveaspects are made in the constant domain CL and the constant domain CH1of the second (and, where present, the third) antigen binding domain,the constant domain CL of the second (and, where present, the third)antigen binding domain is of kappa isotype.

Alternatively, the amino acid substitutions according to the aboveaspects may be made in the constant domain CL and the constant domainCH1 of the first antigen binding domain instead of in the constantdomain CL and the constant domain CH1 of the second (and, where present,the third) antigen binding domain. In preferred such aspects, theconstant domain CL of the first antigen binding domain is of kappaisotype.

Accordingly, in one aspect, in the constant domain CL of the firstantigen binding domain the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first antigenbinding domain the amino acid at position 147 or the amino acid atposition 213 is substituted independently by glutamic acid (E), oraspartic acid (D) (numbering according to Kabat EU index). In a furtheraspect, in the constant domain CL of the first antigen binding domainthe amino acid at position 124 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat), andin the constant domain CH1 of the first antigen binding domain the aminoacid at position 147 is substituted independently by glutamic acid (E),or aspartic acid (D) (numbering according to Kabat EU index).

In still another aspect, in the constant domain CL of the first antigenbinding domain the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first antigenbinding domain the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In one aspect, in the constant domain CL of the first antigen bindingdomain the amino acid at position 124 is substituted by lysine (K)(numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) (numbering according to Kabat), and in theconstant domain CH1 of the first antigen binding domain the amino acidat position 147 is substituted by glutamic acid (E) (numbering accordingto Kabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index).

In another aspect, in the constant domain CL of the first antigenbinding domain the amino acid at position 124 is substituted by lysine(K) (numbering according to Kabat) and the amino acid at position 123 issubstituted by arginine (R) (numbering according to Kabat), and in theconstant domain CH1 of the first antigen binding domain the amino acidat position 147 is substituted by glutamic acid (E) (numbering accordingto Kabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index).

In a preferred aspect, the (multispecific) antibody of the inventioncomprises

(a) a first antigen binding domain that binds to CD3, wherein the firstantigen binding domain is a Fab molecule wherein the variable domains VLand VH of the Fab light chain and the Fab heavy chain are replaced byeach other, and comprises a heavy chain variable region (VH) comprisinga heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 2,a HCDR 2 of SEQ ID NO: 3, and a HCDR 3 of SEQ ID NO: 5, and a lightchain variable region (VL) comprising a light chain complementaritydetermining region (LCDR) 1 of SEQ ID NO: 8, a LCDR 2 of SEQ ID NO: 9and a LCDR 3 of SEQ ID NO: 10, and(b) a second and optionally a third antigen binding domain that bindsCD19;wherein in the constant domain CL of the second (and, where present, thethird) antigen binding domain the amino acid at position 124 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat) (in a preferred aspect independently bylysine (K) or arginine (R)) and the amino acid at position 123 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat) (in a preferred aspect independently bylysine (K) or arginine (R)), and in the constant domain CH1 of thesecond (and, where present, third) antigen binding domain the amino acidat position 147 is substituted independently by glutamic acid (E), oraspartic acid (D) (numbering according to Kabat EU index) and the aminoacid at position 213 is substituted independently by glutamic acid (E),or aspartic acid (D) (numbering according to Kabat EU index).

d) Multispecific Antibody Formats

The (multispecific) antibody according to the present invention can havea variety of configurations. Exemplary configurations are depicted inFIG. 1.

In preferred aspects, the antigen binding domains comprised in the(multispecific) antibody are Fab molecules. In such aspects, the first,second, third etc. antigen binding domain may be referred to herein asfirst, second, third etc. Fab molecule, respectively.

In one aspect, the first and the second antigen binding domain of the(multispecific) antibody are fused to each other, optionally via apeptide linker. In preferred aspects, the first and the second antigenbinding domain are each a Fab molecule. In one such aspect, the firstantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingdomain. In another such aspect, the second antigen binding domain isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first antigen binding domain. In aspects whereineither (i) the first antigen binding domain is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding domain or (ii) the second antigen binding domainis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the first antigen binding domain, additionallythe Fab light chain of the first antigen binding domain and the Fablight chain of the second antigen binding domain may be fused to eachother, optionally via a peptide linker.

A (multispecific) antibody with a single antigen binding domain (such asa Fab molecule) capable of specific binding to a second antigen, e.g. atarget cell antigen such as CD19, (for example as shown in FIG. 1A, D,G, H, K, L) is useful, particularly in cases where internalization ofthe second antigen is to be expected following binding of a highaffinity antigen binding domain. In such cases, the presence of morethan one antigen binding domain specific for the second antigen mayenhance internalization of the second antigen, thereby reducing itsavailability.

In other cases, however, it will be advantageous to have a(multispecific) antibody comprising two or more antigen binding domains(such as Fab molecules) specific for a second antigen, e.g. a targetcell antigen such as CD19 (see examples shown in FIG. 1B, 1C, 1E, 1F,1I, 1J, 1M or 1N), for example to optimize targeting to the target siteor to allow crosslinking of target cell antigens.

Accordingly, in preferred aspects, the (multispecific) antibodyaccording to the present invention comprises a third antigen bindingdomain.

In one aspect, the third antigen binding domain binds to CD19. In oneaspect, the third antigen binding domain is a Fab molecule.

In one aspect, the third antigen domain is identical to the secondantigen binding domain.

In some aspects, the third and the second antigen binding domain areeach a Fab molecule and the third antigen binding domain is identical tothe second antigen binding domain. Thus, in these aspects, the secondand the third antigen binding domain comprise the same heavy and lightchain amino acid sequences and have the same arrangement of domains(i.e. conventional or crossover). Furthermore, in these aspects, thethird antigen binding domain comprises the same amino acidsubstitutions, if any, as the second antigen binding domain. Forexample, the amino acid substitutions described herein as “chargemodifications” will be made in the constant domain CL and the constantdomain CH1 of each of the second antigen binding domain and the thirdantigen binding domain. Alternatively, said amino acid substitutions maybe made in the constant domain CL and the constant domain CH1 of thefirst antigen binding domain (which in preferred aspects is also a Fabmolecule), but not in the constant domain CL and the constant domain CH1of the second antigen binding domain and the third antigen bindingdomain.

Like the second antigen binding domain, the third antigen binding domainpreferably is a conventional Fab molecule. Aspects wherein the secondand the third antigen binding domains are crossover Fab molecules (andthe first antigen binding domain is a conventional Fab molecule) are,however, also contemplated. Thus, in preferred aspects, the second andthe third antigen binding domains are each a conventional Fab molecule,and the first antigen binding domain is a crossover Fab molecule asdescribed herein, i.e. a Fab molecule wherein the variable domains VHand VL or the constant domains CL and CH1 of the Fab heavy and lightchains are exchanged/replaced by each other. In other aspects, thesecond and the third antigen binding domains are each a crossover Fabmolecule and the first antigen binding domain is a conventional Fabmolecule. If a third antigen binding domain is present, in a preferredaspect the first antigen domain binds to CD3, and the second and thirdantigen binding domain bind to CD19.

In preferred aspects, the (multispecific) antibody of the inventioncomprises an Fc domain composed of a first and a second subunit. Thefirst and the second subunit of the Fc domain are capable of stableassociation.

The (multispecific) antibody according to the invention can havedifferent configurations, i.e. the first, second (and optionally third)antigen binding domain may be fused to each other and to the Fc domainin different ways. The components may be fused to each other directlyor, preferably, via one or more suitable peptide linkers. Where fusionof a Fab molecule is to the N-terminus of a subunit of the Fc domain, itis typically via an immunoglobulin hinge region.

In some aspects, the first and the second antigen binding domain areeach a Fab molecule and the first antigen binding domain is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain. In such aspects, the second antigenbinding domain may be fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the first antigen bindingdomain or to the N-terminus of the other one of the subunits of the Fcdomain. In preferred such aspects, the second antigen binding domain isa conventional Fab molecule, and the first antigen binding domain is acrossover Fab molecule as described herein, i.e. a Fab molecule whereinthe variable domains VH and VL or the constant domains CL and CH1 of theFab heavy and light chains are exchanged/replaced by each other. Inother such aspects, the second antigen binding domain is a crossover Fabmolecule and the first antigen binding domain is a conventional Fabmolecule.

In one aspect, the first and the second antigen binding domain are eacha Fab molecule, the first antigen binding domain is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain, and the second antigen binding domainis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the first antigen binding domain. In a specificaspect, the (multispecific) antibody essentially consists of the firstand the second Fab molecule, the Fc domain composed of a first and asecond subunit, and optionally one or more peptide linkers, wherein thesecond Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the first Fab molecule, and thefirst Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the first or the second subunit of the Fc domain. Sucha configuration is schematically depicted in FIGS. 1G and 1K (with thefirst antigen binding domain in these examples being a VH/VL crossoverFab molecule). Optionally, the Fab light chain of the first Fab moleculeand the Fab light chain of the second Fab molecule may additionally befused to each other.

In another aspect, the first and the second antigen binding domain areeach a Fab molecule and the first and the second antigen binding domainare each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain. In a specificaspect, the (multispecific) antibody essentially consists of the firstand the second Fab molecule, the Fc domain composed of a first and asecond subunit, and optionally one or more peptide linkers, wherein thefirst and the second Fab molecule are each fused at the C-terminus ofthe Fab heavy chain to the N-terminus of one of the subunits of the Fcdomain. Such a configuration is schematically depicted in FIGS. 1A and1D (in these examples with the first antigen binding domain being aVH/VL crossover Fab molecule and the second antigen binding domain beinga conventional Fab molecule). The first and the second Fab molecule maybe fused to the Fc domain directly or through a peptide linker. In apreferred aspect the first and the second Fab molecule are each fused tothe Fc domain through an immunoglobulin hinge region. In a specificaspect, the immunoglobulin hinge region is a human IgG₁ hinge region,particularly where the Fc domain is an IgG₁ Fc domain.

In some aspects, the first and the second antigen binding domain areeach a Fab molecule and the second antigen binding domain is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the first orthe second subunit of the Fc domain. In such aspects, the first antigenbinding domain may be fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the first antigen bindingdomain or (as described above) to the N-terminus of the other one of thesubunits of the Fc domain. In preferred such aspects, said secondantigen binding domain is a conventional Fab molecule, and the firstantigen binding domain is a crossover Fab molecule as described herein,i.e. a Fab molecule wherein the variable domains VH and VL or theconstant domains CL and CH1 of the Fab heavy and light chains areexchanged/replaced by each other. In other such aspects, said secondantigen binding domain is a crossover Fab molecule and the first antigenbinding domain is a conventional Fab molecule.

In one aspect, the first and the second antigen binding domain are eacha Fab molecule, the second antigen binding domain is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain, and the first antigen binding domain isfused at the

C-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second antigen binding domain. In a specific aspect, the(multispecific) antibody essentially consists of the first and thesecond Fab molecule, the Fc domain composed of a first and a secondsubunit, and optionally one or more peptide linkers, wherein the firstFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second Fab molecule, and thesecond Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the first or the second subunit of the Fc domain. Sucha configuration is schematically depicted in FIGS. 1H and 1L (in theseexamples with the first antigen binding domain being a VH/VL crossoverFab molecule and the second antigen binding domain being a conventionalFab molecule). Optionally, the Fab light chain of the first Fab moleculeand the Fab light chain of the second Fab molecule may additionally befused to each other.

In some aspects, a third antigen binding domain, particularly a thirdFab molecule, is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first or second subunit of the Fc domain. In preferredsuch aspects, said second and third antigen binding domains are each aconventional Fab molecule, and the first antigen binding domain is acrossover Fab molecule as described herein, i.e. a Fab molecule whereinthe variable domains VH and VL or the constant domains CL and CH1 of theFab heavy and light chains are exchanged/replaced by each other. Inother such aspects, said second and third antigen binding domains areeach a crossover Fab molecule and the first antigen binding domain is aconventional Fab molecule.

In a preferred such aspect, the first and the third antigen bindingdomain are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain, and the secondantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first Fab molecule. In aspecific aspect, the (multispecific) antibody essentially consists ofthe first, the second and the third Fab molecule, the Fc domain composedof a first and a second subunit, and optionally one or more peptidelinkers, wherein the second Fab molecule is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule, and the first Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first subunitof the Fc domain, and wherein the third Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the secondsubunit of the Fc domain. Such a configuration is schematically depictedin FIGS. 1B and 1E (in these examples with the first antigen bindingdomain being a VH/VL crossover Fab molecule, and the second and thethird antigen binding domain being a conventional Fab molecule), andFIGS. 1J and 1N (in these examples with the first antigen binding domainbeing a conventional Fab molecule, and the second and the third antigenbinding domain being a VH/VL crossover Fab molecule). The first and thethird Fab molecule may be fused to the Fc domain directly or through apeptide linker. In a preferred aspect, the first and the third Fabmolecule are each fused to the Fc domain through an immunoglobulin hingeregion. In a specific aspect, the immunoglobulin hinge region is a humanIgG₁ hinge region, particularly where the Fc domain is an IgG₁ Fcdomain. Optionally, the Fab light chain of the first Fab molecule andthe Fab light chain of the second Fab molecule may additionally be fusedto each other.

In another such aspect, the second and the third antigen binding domainare each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain, and the firstantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingdomain. In a specific aspect, the (multispecific) antibody essentiallyconsists of the first, the second and the third Fab molecule, the Fcdomain composed of a first and a second subunit, and optionally one ormore peptide linkers, wherein the first Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second Fab molecule, and the second Fab molecule is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain, and wherein the third Fab molecule is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the secondsubunit of the Fc domain. Such a configuration is schematically depictedin FIGS. 1C and 1F (in these examples with the first antigen bindingdomain being a VH/VL crossover Fab molecule, and the second and thethird antigen binding domain being a conventional Fab molecule) and inFIGS. 1I and 1M (in these examples with the first antigen binding domainbeing a conventional Fab molecule, and the second and the third antigenbinding domain being a VH/VL crossover Fab molecule). The second and thethird Fab molecule may be fused to the Fc domain directly or through apeptide linker. In a preferred aspect the second and the third Fabmolecule are each fused to the Fc domain through an immunoglobulin hingeregion. In a specific aspect, the immunoglobulin hinge region is a humanIgG₁ hinge region, particularly where the Fc domain is an IgG₁ Fcdomain. Optionally, the Fab light chain of the first Fab molecule andthe Fab light chain of the second Fab molecule may additionally be fusedto each other.

In configurations of the (multispecific) antibody wherein a Fab moleculeis fused at the C-terminus of the Fab heavy chain to the N-terminus ofeach of the subunits of the Fc domain through an immunoglobulin hingeregion, the two Fab molecules, the hinge regions and the Fc domainessentially form an immunoglobulin molecule. In a preferred aspect theimmunoglobulin molecule is an IgG class immunoglobulin. In an even morepreferred aspect the immunoglobulin is an IgG₁ subclass immunoglobulin.In another aspect the immunoglobulin is an IgG₄ subclass immunoglobulin.In a further preferred aspect the immunoglobulin is a humanimmunoglobulin. In other aspects the immunoglobulin is a chimericimmunoglobulin or a humanized immunoglobulin. In one aspect, theimmunoglobulin comprises a human constant region, particularly a humanFc region.

In some of the (multispecific) antibodies of the invention, the Fablight chain of the first Fab molecule and the Fab light chain of thesecond Fab molecule are fused to each other, optionally via a peptidelinker. Depending on the configuration of the first and the second Fabmolecule, the Fab light chain of the first Fab molecule may be fused atits C-terminus to the N-terminus of the Fab light chain of the secondFab molecule, or the Fab light chain of the second Fab molecule may befused at its C-terminus to the N-terminus of the Fab light chain of thefirst Fab molecule. Fusion of the Fab light chains of the first and thesecond Fab molecule further reduces mispairing of unmatched Fab heavyand light chains, and also reduces the number of plasmids needed forexpression of some of the (multispecific) antibody of the invention.

The antigen binding domains may be fused to the Fc domain or to eachother directly or through a peptide linker, comprising one or more aminoacids, typically about 2-20 amino acids. Peptide linkers are known inthe art and are described herein. Suitable, non-immunogenic peptidelinkers include, for example, (G₄S)_(n), (SG₄)_(n), (G₄S)_(n),G₄(SG₄)_(n) or (G₄S)_(n)G5 peptide linkers. “n” is generally an integerfrom 1 to 10, typically from 2 to 4. In one aspect said peptide linkerhas a length of at least 5 amino acids, in one aspect a length of 5 to100, in a further aspect of 10 to 50 amino acids. In one aspect saidpeptide linker is (GxS)_(n) or (GxS)_(n)G_(m) with G=glycine, S=serine,and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=1, 2, 3, 4 or 5and m=0, 1, 2, 3, 4 or 5), in one aspect x=4 and n=2 or 3, in a furtheraspect x=4 and n=2, in yet a further aspect x=4, n=1 and m=5. In oneaspect said peptide linker is (G₄S)₂. In another aspect, said peptidelinker is G₄SG₅. A particularly suitable peptide linker for fusing theFab light chains of the first and the second Fab molecule to each otheris (G₄S)₂. An exemplary peptide linker suitable for connecting the Fabheavy chains of the first and the second Fab fragments comprises thesequence (D)-(G₄S)₂ (SEQ ID NOs 48 and 49). Another particularlysuitable such linker comprises the sequence (D)-G₄SG₅ (SEQ ID NOs 50 and51). Additionally, linkers may comprise (a portion of) an immunoglobulinhinge region. Particularly where a Fab molecule is fused to theN-terminus of an Fc domain subunit, it may be fused via animmunoglobulin hinge region or a portion thereof, with or without anadditional peptide linker.

In certain aspects the (multispecific) antibody according to theinvention comprises a polypeptide wherein the Fab light chain variableregion of the first Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the first Fab molecule (i.e.the first Fab molecule comprises a crossover Fab heavy chain, whereinthe heavy chain variable region is replaced by a light chain variableregion), which in turn shares a carboxy-terminal peptide bond with an Fcdomain subunit (VL₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)), and a polypeptide whereinthe Fab heavy chain of the second Fab molecule shares a carboxy-terminalpeptide bond with an Fc domain subunit (VH₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)). Insome aspects the (multispecific) antibody further comprises apolypeptide wherein the Fab heavy chain variable region of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab light chainconstant region of the first Fab molecule (VH₍₁₎-CL₍₁₎) and the Fablight chain polypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). Incertain aspects the polypeptides are covalently linked, e.g., by adisulfide bond. In certain aspects the (multispecific) antibodyaccording to the invention comprises a polypeptide wherein the Fab heavychain variable region of the first Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the first Fab molecule (i.e. the first Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with an Fc domain subunit(VH₍₁₎-CL₍₁₎-CH2-CH3(-CH4)), and a polypeptide wherein the Fab heavychain of the second Fab molecule shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)). In some aspectsthe (multispecific) antibody further comprises a polypeptide wherein theFab light chain variable region of the first Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the first Fab molecule (VL₍₁₎-CH1₍₁₎) and the Fab light chainpolypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). In certain aspectsthe polypeptides are covalently linked, e.g., by a disulfide bond. Insome aspects, the (multispecific) antibody comprises a polypeptidewherein the Fab light chain variable region of the first Fab moleculeshares a carboxy-terminal peptide bond with the Fab heavy chain constantregion of the first Fab molecule (i.e. the first Fab molecule comprisesa crossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the second Fabmolecule, which in turn shares a carboxy-terminal peptide bond with anFc domain subunit (VL₍₁₎-CH1₍₁₎-VH₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)). In otheraspects, the (multispecific) antibody comprises a polypeptide whereinthe Fab heavy chain of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain variable region of the first Fabmolecule which in turn shares a carboxy-terminal peptide bond with theFab heavy chain constant region of the first Fab molecule (i.e. thefirst Fab molecule comprises a crossover Fab heavy chain, wherein theheavy chain variable region is replaced by a light chain variableregion), which in turn shares a carboxy-terminal peptide bond with an Fcdomain subunit (VH₍₂₎-CH1₍₂₎-VL₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In some ofthese aspects the (multispecific) antibody further comprises a crossoverFab light chain polypeptide of the first Fab molecule, wherein the Fabheavy chain variable region of the first Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the first Fab molecule (VH₍₁₎-CL₍₁₎), and the Fab light chainpolypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). In others of theseaspects the (multispecific) antibody further comprises a polypeptidewherein the Fab heavy chain variable region of the first Fab moleculeshares a carboxy-terminal peptide bond with the Fab light chain constantregion of the first Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain polypeptide of the second Fabmolecule (VH₍₁₎-CL₍₁₎-VL₍₂₎-CL₍₂₎), or a polypeptide wherein the Fablight chain polypeptide of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain variable regionof the first Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the first Fabmolecule (VL₍₂₎-CL₍₂₎-VH₍₁₎-CL₍₁₎), as appropriate. The (multispecific)antibody according to these aspects may further comprise (i) an Fcdomain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptidewherein the Fab heavy chain of a third Fab molecule shares acarboxy-terminal peptide bond with an Fc domain subunit(VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) and the Fab light chain polypeptide of athird Fab molecule (VL₍₃₎-CL₍₃₎). In certain aspects the polypeptidesare covalently linked, e.g., by a disulfide bond.

In some aspects, the (multispecific) antibody comprises a polypeptidewherein the Fab heavy chain variable region of the first Fab moleculeshares a carboxy-terminal peptide bond with the Fab light chain constantregion of the first Fab molecule (i.e. the first Fab molecule comprisesa crossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the second Fabmolecule, which in turn shares a carboxy-terminal peptide bond with anFc domain subunit (VH₍₁₎-CL₍₁₎-VH₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)). In otheraspects, the (multispecific) antibody comprises a polypeptide whereinthe Fab heavy chain of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain variable region of the first Fabmolecule which in turn shares a carboxy-terminal peptide bond with theFab light chain constant region of the first Fab molecule (i.e. thefirst Fab molecule comprises a crossover Fab heavy chain, wherein theheavy chain constant region is replaced by a light chain constantregion), which in turn shares a carboxy-terminal peptide bond with an Fcdomain subunit (VH₍₂₎-CH1₍₂₎-VH₍₁₎-CL₍₁₎-CH2-CH3(-CH4)). In some ofthese aspects the (multispecific) antibody further comprises a crossoverFab light chain polypeptide of the first Fab molecule, wherein the Fablight chain variable region of the first Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the first Fab molecule (VL₍₁₎-CH1₍₁₎), and the Fab light chainpolypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). In others of theseaspects the (multispecific) antibody further comprises a polypeptidewherein the Fab light chain variable region of the first Fab moleculeshares a carboxy-terminal peptide bond with the Fab heavy chain constantregion of the first Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain polypeptide of the second Fabmolecule (VL₍₁₎-CH1₍₁₎-VL₍₂₎-CL₍₂₎), or a polypeptide wherein the Fablight chain polypeptide of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain variable regionof the first Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the first Fabmolecule (VL₍₂₎-CL₍₂₎-VH₍₁₎-CL₍₁₎), as appropriate. The (multispecific)antibody according to these aspects may further comprise (i) an Fcdomain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptidewherein the Fab heavy chain of a third Fab molecule shares acarboxy-terminal peptide bond with an Fc domain subunit(VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) and the Fab light chain polypeptide of athird Fab molecule (VL₍₃₎-CL₍₃₎). In certain aspects the polypeptidesare covalently linked, e.g., by a disulfide bond.

In certain aspects, the (multispecific) antibody does not comprise an Fcdomain. In preferred such aspects, said second and, if present, thirdantigen binding domains are each a conventional Fab molecule, and thefirst antigen binding domain is a crossover Fab molecule as describedherein, i.e. a Fab molecule wherein the variable domains VH and VL orthe constant domains CL and CH1 of the Fab heavy and light chains areexchanged/replaced by each other. In other such aspects, said secondand, if present, third antigen binding domains are each a crossover Fabmolecule and the first antigen binding domain is a conventional Fabmolecule.

In one such aspect, the (multispecific) antibody essentially consists ofthe first and the second antigen binding domain, and optionally one ormore peptide linkers, wherein the first and the second antigen bindingdomain are both Fab molecules and the second antigen binding domain isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first antigen binding domain. Such aconfiguration is schematically depicted in FIGS. 1O and 1S (in theseexamples with the first antigen binding domain being a VH/VL crossoverFab molecule and the second antigen binding domain being a conventionalFab molecule).

In another such aspect, the (multispecific) antibody essentiallyconsists of the first and the second antigen binding domain, andoptionally one or more peptide linkers, wherein the first and the secondantigen binding domain are both Fab molecules and the first antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second antigen binding domain.Such a configuration is schematically depicted in FIGS. 1P and 1T (inthese examples with the first antigen binding domain being a VH/VLcrossover Fab molecule and the second antigen binding domain being aconventional Fab molecule).

In some aspects, the second Fab molecule is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule, and the (multispecific) antibody further comprises athird antigen binding domain, particularly a third Fab molecule, whereinsaid third Fab molecule is fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the second Fabmolecule. In certain such aspects, the (multispecific) antibodyessentially consists of the first, the second and the third Fabmolecule, and optionally one or more peptide linkers, wherein the secondFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first Fab molecule, and thethird Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the second Fab molecule. Such aconfiguration is schematically depicted in FIGS. 1Q and 1U (in theseexamples with the first antigen binding domain being a VH/VL crossoverFab molecule and the second and the third antigen binding domain eachbeing a conventional Fab molecule), or FIGS. 1X and 1Z (in theseexamples with the first antigen binding domain being a conventional Fabmolecule and the second and the third antigen binding domain each beinga VH/VL crossover Fab molecule).

In some aspects, the first Fab molecule is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule, and the (multispecific) antibody further comprisesa third antigen binding domain, particularly a third Fab molecule,wherein said third Fab molecule is fused at the N-terminus of the Fabheavy chain to the C-terminus of the Fab heavy chain of the second Fabmolecule. In certain such aspects, the (multispecific) antibodyessentially consists of the first, the second and the third Fabmolecule, and optionally one or more peptide linkers, wherein the firstFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second Fab molecule, and thethird Fab molecule is fused at the N-terminus of the Fab heavy chain tothe C-terminus of the Fab heavy chain of the second Fab molecule. Such aconfiguration is schematically depicted in FIGS. 1R and 1V (in theseexamples with the first antigen binding domain being a VH/VL crossoverFab molecule and the second and the third antigen binding domain eachbeing a conventional Fab molecule), or FIGS. 1W and 1Y (in theseexamples with the first antigen binding domain being a conventional Fabmolecule and the second and the third antigen binding domain each beinga VH/VL crossover Fab molecule).

In certain aspects the (multispecific) antibody according to theinvention comprises a polypeptide wherein the Fab heavy chain of thesecond Fab molecule shares a carboxy-terminal peptide bond with the Fablight chain variable region of the first Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain constantregion of the first Fab molecule (i.e. the first Fab molecule comprisesa crossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region) (VH₍₂₎-CH1₍₂₎-VL₍₁₎-CH1₍₁₎).In some aspects the (multispecific) antibody further comprises apolypeptide wherein the Fab heavy chain variable region of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab light chainconstant region of the first Fab molecule (VH₍₁₎-CL₍₁₎) and the Fablight chain polypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎).

In certain aspects the (multispecific) antibody according to theinvention comprises a polypeptide wherein the Fab light chain variableregion of the first Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the first Fab molecule (i.e.the first Fab molecule comprises a crossover Fab heavy chain, whereinthe heavy chain variable region is replaced by a light chain variableregion), which in turn shares a carboxy-terminal peptide bond with theFab heavy chain of the second Fab molecule (VL₍₁₎-CH1₍₁₎-VH₍₂₎-CH1₍₂₎).In some aspects the (multispecific) antibody further comprises apolypeptide wherein the Fab heavy chain variable region of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab light chainconstant region of the first Fab molecule (VH₍₁₎-CL₍₁₎) and the Fablight chain polypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎).

In certain aspects the (multispecific) antibody according to theinvention comprises a polypeptide wherein the Fab heavy chain of thesecond Fab molecule shares a carboxy-terminal peptide bond with the Fabheavy chain variable region of the first Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab light chain constantregion of the first Fab molecule (i.e. the first Fab molecule comprisesa crossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region) (VH₍₂₎-CH1₍₂₎-VH₍₁₎-CL₍₁₎. Insome aspects the (multispecific) antibody further comprises apolypeptide wherein the Fab light chain variable region of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab heavy chainconstant region of the first Fab molecule (VL₍₁₎-CH1₍₁₎) and the Fablight chain polypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎).

In certain aspects the (multispecific) antibody according to theinvention comprises a polypeptide wherein the Fab heavy chain variableregion of the first Fab molecule shares a carboxy-terminal peptide bondwith the Fab light chain constant region of the first Fab molecule (i.e.the first Fab molecule comprises a crossover Fab heavy chain, whereinthe heavy chain constant region is replaced by a light chain constantregion), which in turn shares a carboxy-terminal peptide bond with theFab heavy chain of the second Fab molecule (VH₍₁₎-CL₍₁₎-VH₍₂₎-CH1₍₂₎).In some aspects the (multispecific) antibody further comprises apolypeptide wherein the Fab light chain variable region of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab heavy chainconstant region of the first Fab molecule (VL₍₁₎-CH1₍₁₎) and the Fablight chain polypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎).

In certain aspects the (multispecific) antibody according to theinvention comprises a polypeptide wherein the Fab heavy chain of a thirdFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain of the second Fab molecule, which in turn shares acarboxy-terminal peptide bond with the Fab light chain variable regionof the first Fab molecule, which in turn shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the first Fabmolecule (i.e. the first Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region) (VH₍₃₎-CH1₍₃₎-VH₍₂₎-CH1₍₂₎-VL₍₁₎-CH1₍₁₎). In someaspects the (multispecific) antibody further comprises a polypeptidewherein the Fab heavy chain variable region of the first Fab moleculeshares a carboxy-terminal peptide bond with the Fab light chain constantregion of the first Fab molecule (VH₍₁₎-CL₍₁₎) and the Fab light chainpolypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). In some aspectsthe (multispecific) antibody further comprises the Fab light chainpolypeptide of a third Fab molecule (VL₍₃₎-CL₍₃₎). In certain aspectsthe (multispecific) antibody according to the invention comprises apolypeptide wherein the Fab heavy chain of a third Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain of the second Fabmolecule, which in turn shares a carboxy-terminal peptide bond with theFab heavy chain variable region of the first Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab light chain constantregion of the first Fab molecule (i.e. the first Fab molecule comprisesa crossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region)(VH₍₃₎-CH1₍₃₎-VH₍₂₎-CH1₍₂₎-VH₍₁₎-CL₍₁₎). In some aspects the(multispecific) antibody further comprises a polypeptide wherein the Fablight chain variable region of the first Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the first Fab molecule (VL₍₁₎-CH1₍₁₎) and the Fab light chainpolypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). In some aspectsthe (multispecific) antibody further comprises the Fab light chainpolypeptide of a third Fab molecule (VL₍₃₎-CL₍₃₎). In certain aspectsthe (multispecific) antibody according to the invention comprises apolypeptide wherein the Fab light chain variable region of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab heavy chainconstant region of the first Fab molecule (i.e. the first Fab moleculecomprises a crossover Fab heavy chain, wherein the heavy chain variableregion is replaced by a light chain variable region), which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain of thesecond Fab molecule, which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of a third Fab molecule(VL₍₁₎-CH1₍₁₎-VH₍₂₎-CH1₍₂₎-VH₍₃₎-CH1₍₃₎). In some aspects the(multispecific) antibody further comprises a polypeptide wherein the Fabheavy chain variable region of the first Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the first Fab molecule (VH₍₁₎-CL₍₁₎) and the Fab light chainpolypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). In some aspectsthe (multispecific) antibody further comprises the Fab light chainpolypeptide of a third Fab molecule (VL₍₃₎-CL₍₃₎). In certain aspectsthe (multispecific) antibody according to the invention comprises apolypeptide wherein the Fab heavy chain variable region of the first Fabmolecule shares a carboxy-terminal peptide bond with the Fab light chainconstant region of the first Fab molecule (i.e. the first Fab moleculecomprises a crossover Fab heavy chain, wherein the heavy chain constantregion is replaced by a light chain constant region), which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain of thesecond Fab molecule, which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of a third Fab molecule(VH₍₁₎-CL₍₁₎-VH₍₂₎-CH1₍₂₎-VH₍₃₎-CH1₍₃₎). In some aspects the(multispecific) antibody further comprises a polypeptide wherein the Fablight chain variable region of the first Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the first Fab molecule (VL₍₁₎-CH1₍₁₎) and the Fab light chainpolypeptide of the second Fab molecule (VL₍₂₎-CL₍₂₎). In some aspectsthe (multispecific) antibody further comprises the Fab light chainpolypeptide of a third Fab molecule (VL₍₃₎-CL₍₃₎). In certain aspectsthe (multispecific) antibody according to the invention comprises apolypeptide wherein the Fab heavy chain of the first Fab molecule sharesa carboxy-terminal peptide bond with the Fab light chain variable regionof the second Fab molecule, which in turn shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with the Fab light chain variable region of a third Fab molecule,which in turn shares a carboxy-terminal peptide bond with the Fab heavychain constant region of a third Fab molecule (i.e. the third Fabmolecule comprises a crossover Fab heavy chain, wherein the heavy chainvariable region is replaced by a light chain variable region)(VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎-VL₍₃₎-CH1₍₃₎). In some aspects the(multispecific) antibody further comprises a polypeptide wherein the Fabheavy chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (VH₍₂₎-CL₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In some aspects the(multispecific) antibody further comprises a polypeptide wherein the Fabheavy chain variable region of a third Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof a third Fab molecule (VH₍₃₎-CL₍₃₎).

In certain aspects the (multispecific) antibody according to theinvention comprises a polypeptide wherein the Fab heavy chain of thefirst Fab molecule shares a carboxy-terminal peptide bond with the Fabheavy chain variable region of the second Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab light chain constantregion of the second Fab molecule (i.e. the second Fab moleculecomprises a crossover Fab heavy chain, wherein the heavy chain constantregion is replaced by a light chain constant region), which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain variableregion of a third Fab molecule, which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of a third Fabmolecule (i.e. the third Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region) (VH₍₁₎-CH1₍₁₎-VH₍₂₎-CL₍₂₎-VH₍₃₎-CL₍₃₎). In someaspects the (multispecific) antibody further comprises a polypeptidewherein the Fab light chain variable region of the second Fab moleculeshares a carboxy-terminal peptide bond with the Fab heavy chain constantregion of the second Fab molecule (VL₍₂₎-CH1₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In some aspects the(multispecific) antibody further comprises a polypeptide wherein the Fablight chain variable region of a third Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof a third Fab molecule (VL₍₃₎-CH1₍₃₎).

In certain aspects the (multispecific) antibody according to theinvention comprises a polypeptide wherein the Fab light chain variableregion of a third Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of a third Fab molecule (i.e.the third Fab molecule comprises a crossover Fab heavy chain, whereinthe heavy chain variable region is replaced by a light chain variableregion), which in turn shares a carboxy-terminal peptide bond with theFab light chain variable region of the second Fab molecule, which inturn shares a carboxy-terminal peptide bond with the Fab heavy chainconstant region of the second Fab molecule (i.e. the second Fab moleculecomprises a crossover Fab heavy chain, wherein the heavy chain variableregion is replaced by a light chain variable region), which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain of thefirst Fab molecule (VL₍₃₎-CH1₍₃₎-VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎). In someaspects the (multispecific) antibody further comprises a polypeptidewherein the Fab heavy chain variable region of the second Fab moleculeshares a carboxy-terminal peptide bond with the Fab light chain constantregion of the second Fab molecule (VH₍₂₎-CL₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In some aspects the(multispecific) antibody further comprises a polypeptide wherein the Fabheavy chain variable region of a third Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof a third Fab molecule (VH₍₃₎-CL₍₃₎).

In certain aspects the (multispecific) antibody according to theinvention comprises a polypeptide wherein the Fab heavy chain variableregion of a third Fab molecule shares a carboxy-terminal peptide bondwith the Fab light chain constant region of a third Fab molecule (i.e.the third Fab molecule comprises a crossover Fab heavy chain, whereinthe heavy chain constant region is replaced by a light chain constantregion), which in turn shares a carboxy-terminal peptide bond with theFab heavy chain variable region of the second Fab molecule, which inturn shares a carboxy-terminal peptide bond with the Fab light chainconstant region of the second Fab molecule (i.e. the second Fab moleculecomprises a crossover Fab heavy chain, wherein the heavy chain constantregion is replaced by a light chain constant region), which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain of thefirst Fab molecule (VH₍₃₎-CL₍₃₎-VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎). In someaspects the (multispecific) antibody further comprises a polypeptidewherein the Fab light chain variable region of the second Fab moleculeshares a carboxy-terminal peptide bond with the Fab heavy chain constantregion of the second Fab molecule (VL₍₂₎-CH1₍₂₎) and the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). In some aspects the(multispecific) antibody further comprises a polypeptide wherein the Fablight chain variable region of a third Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof a third Fab molecule (VL₍₃₎-CH1₍₃₎).

In one aspect, the invention provides a (multispecific) antibodycomprising

a) a first antigen binding domain that binds to CD3, wherein the firstantigen binding domain is a Fab molecule wherein the variable domains VLand VH or the constant domains CL and CH1 of the Fab light chain and theFab heavy chain are replaced by each other, and comprises a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 2, a HCDR 2 of SEQ ID NO: 3, and a HCDR 3of SEQ ID NO: 5, and a light chain variable region (VL) comprising alight chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8,a LCDR 2 of SEQ ID NO: 9 and a LCDR 3 of SEQ ID NO: 10;b) a second antigen binding domain that binds to CD19, wherein thesecond antigen binding domain is a (conventional) Fab molecule;c) an Fc domain composed of a first and a second subunit;wherein(i) the first antigen binding domain under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding domain under b), and the second antigen bindingdomain under b) is fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under c), or(ii) the second antigen binding domain under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding domain under a), and the firstantigen binding domain under a) is fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

In a preferred aspect, the invention provides a (multispecific) antibodycomprising

a) a first antigen binding domain that binds to CD3, wherein the firstantigen binding domain is a Fab molecule wherein the variable domains VLand VH or the constant domains CL and CH1 of the Fab light chain and theFab heavy chain are replaced by each other, and comprises a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 2, a HCDR 2 of SEQ ID NO: 3, and a HCDR 3of SEQ ID NO: 5, and a light chain variable region (VL) comprising alight chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8,a LCDR 2 of SEQ ID NO: 9 and a LCDR 3 of SEQ ID NO: 10;b) a second and a third antigen binding domain that bind to CD19,wherein the second and the third antigen binding domain are each a(conventional) Fab molecule; andc) an Fc domain composed of a first and a second subunit;wherein(i) the first antigen binding domain under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding domain under b), and the second antigen bindingdomain under b) and the third antigen binding domain under b) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under c), or(ii) the second antigen binding domain under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding domain under a), and the firstantigen binding domain under a) and the third antigen binding domainunder b) are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under c).

In another aspect, the invention provides a (multispecific) antibodycomprising

a) a first antigen binding domain that binds to CD3, wherein the firstantigen binding domain is a Fab molecule wherein the variable domains VLand VH or the constant domains CL and CH1 of the Fab light chain and theFab heavy chain are replaced by each other, and comprises a heavy chainvariable region (VH) comprising a heavy chain complementary determiningregion (HCDR) 1 of SEQ ID NO: 2, a HCDR 2 of SEQ ID NO: 3, and a HCDR 3of SEQ ID NO: 5, and a light chain variable region (VL) comprising alight chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8,a LCDR 2 of SEQ ID NO: 9 and a LCDR 3 of SEQ ID NO: 10;b) a second antigen binding domain that binds to CD19, wherein thesecond antigen binding domain is a (conventional) Fab molecule;c) an Fc domain composed of a first and a second subunit;wherein(i) the first antigen binding domain under a) and the second antigenbinding domain under b) are each fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

In all of the different configurations of the (multispecific) antibodyaccording to the invention, the amino acid substitutions (“chargemodifications”) described herein, if present, may either be in the CH1and CL domains of the second and (if present) the third antigen bindingdomain/Fab molecule, or in the CH1 and CL domains of the first antigenbinding domain/Fab molecule. Preferably, they are in the CH1 and CLdomains of the second and (if present) the third antigen bindingdomain/Fab molecule. In accordance with the concept of the invention, ifamino acid substitutions as described herein are made in the second(and, if present, the third) antigen binding domain/Fab molecule, nosuch amino acid substitutions are made in the first antigen bindingdomain/Fab molecule. Conversely, if amino acid substitutions asdescribed herein are made in the first antigen binding domain/Fabmolecule, no such amino acid substitutions are made in the second (and,if present, the third) antigen binding domain/Fab molecule. Amino acidsubstitutions are preferably made in (multispecific) antibodiescomprising a Fab molecule wherein the variable domains VL and VH1 of theFab light chain and the Fab heavy chain are replaced by each other.

In preferred aspects of the (multispecific) antibody according to theinvention, particularly wherein amino acid substitutions as describedherein are made in the second (and, if present, the third) antigenbinding domain/Fab molecule, the constant domain CL of the second (and,if present, the third) Fab molecule is of kappa isotype. In otheraspects of the (multispecific) antibody according to the invention,particularly wherein amino acid substitutions as described herein aremade in the first antigen binding domain/Fab molecule, the constantdomain CL of the first antigen binding domain/Fab molecule is of kappaisotype. In some aspects, the constant domain CL of the second (and, ifpresent, the third) antigen binding domain/Fab molecule and the constantdomain CL of the first antigen binding domain/Fab molecule are of kappaisotype.

In one aspect, the invention provides a (multispecific) antibodycomprising

a) a first antigen binding domain that binds to CD3, wherein the firstantigen binding domain is a Fab molecule wherein the variable domains VLand VH of the Fab light chain and the Fab heavy chain are replaced byeach other, and comprises a heavy chain variable region (VH) comprisinga heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 2,a HCDR 2 of SEQ ID NO: 3, and a HCDR 3 of SEQ ID NO: 5, and a lightchain variable region (VL) comprising a light chain complementaritydetermining region (LCDR) 1 of SEQ ID NO: 8, a LCDR 2 of SEQ ID NO: 9and a LCDR 3 of SEQ ID NO: 10;b) a second antigen binding domain that binds to CD19, wherein thesecond antigen binding domain is a (conventional) Fab molecule;c) an Fc domain composed of a first and a second subunit;wherein in the constant domain CL of the second antigen binding domainunder b) the amino acid at position 124 is substituted by lysine (K)(numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most preferably by arginine (R)), and wherein in the constant domainCH1 of the second antigen binding domain under b) the amino acid atposition 147 is substituted by glutamic acid (E) (numbering according toKabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index); and wherein(i) the first antigen binding domain under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding domain under b), and the second antigen bindingdomain under b) is fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under c), or(ii) the second antigen binding domain under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding domain under a), and the firstantigen binding domain under a) is fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

In a preferred aspect, the invention provides a (multispecific) antibodycomprising

a) a first antigen binding domain that binds to CD3, wherein the firstantigen binding domain is a Fab molecule wherein the variable domains VLand VH of the Fab light chain and the Fab heavy chain are replaced byeach other, and comprises a heavy chain variable region (VH) comprisinga heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 2,a HCDR 2 of SEQ ID NO: 3, and a HCDR 3 of SEQ ID NO: 5, and a lightchain variable region (VL) comprising a light chain complementaritydetermining region (LCDR) 1 of SEQ ID NO: 8, a LCDR 2 of SEQ ID NO: 9and a LCDR 3 of SEQ ID NO: 10;b) a second and a third antigen binding domain that bind to CD19,wherein the second and third antigen binding domain are each a(conventional) Fab molecule; andc) an Fc domain composed of a first and a second subunit;wherein in the constant domain CL of the second antigen binding domainunder b) and the third antigen binding domain under b) the amino acid atposition 124 is substituted by lysine (K) (numbering according to Kabat)and the amino acid at position 123 is substituted by lysine (K) orarginine (R) (numbering according to Kabat) (most preferably by arginine(R)), and wherein in the constant domain CH1 of the second antigenbinding domain under b) and the third antigen binding domain under b)the amino acid at position 147 is substituted by glutamic acid (E)(numbering according to Kabat EU index) and the amino acid at position213 is substituted by glutamic acid (E) (numbering according to Kabat EUindex); andwherein(i) the first antigen binding domain under a) is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding domain under b), and the second antigen bindingdomain under b) and the third antigen binding domain under b) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under c), or(ii) the second antigen binding domain under b) is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding domain under a), and the firstantigen binding domain under a) and the third antigen binding domainunder b) are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under c).

In another aspect, the invention provides a (multispecific) antibodycomprising

a) a first antigen binding domain that binds to CD3, wherein the firstantigen binding domain is a Fab molecule wherein the variable domains VLand VH of the Fab light chain and the Fab heavy chain are replaced byeach other, and comprises a heavy chain variable region (VH) comprisinga heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 2,a HCDR 2 of SEQ ID NO: 3, and a HCDR 3 of SEQ ID NO: 5, and a lightchain variable region (VL) comprising a light chain complementaritydetermining region (LCDR) 1 of SEQ ID NO: 8, a LCDR 2 of SEQ ID NO: 9and a LCDR 3 of SEQ ID NO: 10;b) a second antigen binding domain that binds to CD19, wherein thesecond antigen binding domain is a (conventional) Fab molecule;c) an Fc domain composed of a first and a second subunit;wherein in the constant domain CL of the second antigen binding domainunder b) the amino acid at position 124 is substituted by lysine (K)(numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most preferably by arginine (R)), and wherein in the constant domainCH1 of the second antigen binding domain under b) the amino acid atposition 147 is substituted by glutamic acid (E) (numbering according toKabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index); andwherein the first antigen binding domain under a) and the second antigenbinding domain under b) are each fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder c).

According to any of the above aspects, components of the (multispecific)antibody (e.g. Fab molecules, Fc domain) may be fused directly orthrough various linkers, particularly peptide linkers comprising one ormore amino acids, typically about 2-20 amino acids, that are describedherein or are known in the art. Suitable, non-immunogenic peptidelinkers include, for example, (G₄S)_(n), (SG₄)_(n), (G₄S)_(n),G₄(SG₄)_(n) or (G₄S)_(n)G₅ peptide linkers, wherein n is generally aninteger from 1 to 10, typically from 2 to 4.

In a preferred aspect, the invention provides a (multispecific) antibodycomprising

a) a first antigen binding domain that binds CD3, wherein the firstantigen binding domain is a Fab molecule wherein the variable domains VLand VH of the Fab light chain and the Fab heavy chain are replaced byeach other, and comprises a heavy chain variable region (VH) comprisinga heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 2,a HCDR 2 of SEQ ID NO: 3, and a HCDR 3 of SEQ ID NO: 5, and a lightchain variable region (VL) comprising a light chain complementaritydetermining region (LCDR) 1 of SEQ ID NO: 8, a LCDR 2 of SEQ ID NO: 9and a LCDR 3 of SEQ ID NO: 10;b) a second and a third antigen binding domain that bind to CD19,wherein the second and the third antigen binding domain are each a(conventional) Fab molecule, and comprise a heavy chain variable region(VH) comprising a heavy chain complementary determining region (HCDR) 1of SEQ ID NO: 15, a HCDR 2 of SEQ ID NO: 16, and a HCDR 3 of SEQ ID NO:17, and a light chain variable region (VL) comprising a light chaincomplementarity determining region (LCDR) 1 of SEQ ID NO: 19, a LCDR 2of SEQ ID NO: 20 and a LCDR 3 of SEQ ID NO: 21;c) an Fc domain composed of a first and a second subunit;whereinin the constant domain CL of the second and the third antigen bindingdomain under b) the amino acid at position 124 is substituted by lysine(K) (numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most preferably by arginine (R)), and wherein in the constant domainCH1 of the second and the third antigen binding domain under b) theamino acid at position 147 is substituted by glutamic acid (E)(numbering according to Kabat EU index) and the amino acid at position213 is substituted by glutamic acid (E) (numbering according to Kabat EUindex);and wherein furtherthe second antigen binding domain under b) is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst antigen binding domain under a), and the first antigen bindingdomain under a) and the third antigen binding domain under b) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under c).

In a further preferred aspect, the invention provides a (multispecific)antibody comprising

a) a first antigen binding domain that binds to CD3, wherein the firstantigen binding domain is a Fab molecule wherein the variable domains VLand VH of the Fab light chain and the Fab heavy chain are replaced byeach other, and comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 7 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 11;b) a second and a third antigen binding domain that bind to CD19,wherein the second and the third antigen binding domain are each a(conventional) Fab molecule, and comprise a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 18 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 22;c) an Fc domain composed of a first and a second subunit;whereinin the constant domain CL of the second and the third antigen bindingdomain under b) the amino acid at position 124 is substituted by lysine(K) (numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most preferably by arginine (R)), and wherein in the constant domainCH1 of the second and the third antigen binding domain under b) theamino acid at position 147 is substituted by glutamic acid (E)(numbering according to Kabat EU index) and the amino acid at position213 is substituted by glutamic acid (E) (numbering according to Kabat EUindex);and wherein furtherthe second antigen binding domain under b) is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst antigen binding domain under a), and the first antigen bindingdomain under a) and the third antigen binding domain under b) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under c).

In a further preferred aspect, the invention provides a (multispecific)antibody comprising

a) a first antigen binding domain that binds CD3, wherein the firstantigen binding domain is a Fab molecule wherein the variable domains VLand VH of the Fab light chain and the Fab heavy chain are replaced byeach other, and comprises a heavy chain variable region (VH) comprisinga heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 2,a HCDR 2 of SEQ ID NO: 3, and a HCDR 3 of SEQ ID NO: 5, and a lightchain variable region (VL) comprising a light chain complementaritydetermining region (LCDR) 1 of SEQ ID NO: 8, a LCDR 2 of SEQ ID NO: 9and a LCDR 3 of SEQ ID NO: 10;b) a second and a third antigen binding domain that bind to CD19,wherein the second and the third antigen binding domain are each a(conventional) Fab molecule, and comprise a heavy chain variable region(VH) comprising a heavy chain complementary determining region (HCDR) 1of SEQ ID NO: 28, a HCDR 2 of SEQ ID NO: 29, and a HCDR 3 of SEQ ID NO:30, and a light chain variable region (VL) comprising a light chaincomplementarity determining region (LCDR) 1 of SEQ ID NO: 32, a LCDR 2of SEQ ID NO: 33 and a LCDR 3 of SEQ ID NO: 34;c) an Fc domain composed of a first and a second subunit;whereinin the constant domain CL of the second and the third antigen bindingdomain under b) the amino acid at position 124 is substituted by lysine(K) (numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most preferably by arginine (R)), and wherein in the constant domainCH1 of the second and the third antigen binding domain under b) theamino acid at position 147 is substituted by glutamic acid (E)(numbering according to Kabat EU index) and the amino acid at position213 is substituted by glutamic acid (E) (numbering according to Kabat EUindex);and wherein furtherthe second antigen binding domain under b) is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst antigen binding domain under a), and the first antigen bindingdomain under a) and the third antigen binding domain under b) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under c).

In a yet further preferred aspect, the invention provides a(multispecific) antibody comprising

a) a first antigen binding domain that binds to CD3, wherein the firstantigen binding domain is a Fab molecule wherein the variable domains VLand VH of the Fab light chain and the Fab heavy chain are replaced byeach other, and comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 7 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 11;b) a second and a third antigen binding domain that bind to CD19,wherein the second and the third antigen binding domain are each a(conventional) Fab molecule, and comprise a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 31 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 35;c) an Fc domain composed of a first and a second subunit;whereinin the constant domain CL of the second and the third antigen bindingdomain under b) the amino acid at position 124 is substituted by lysine(K) (numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) or arginine (R) (numbering according to Kabat)(most preferably by arginine (R)), and wherein in the constant domainCH1 of the second and the third antigen binding domain under b) theamino acid at position 147 is substituted by glutamic acid (E)(numbering according to Kabat EU index) and the amino acid at position213 is substituted by glutamic acid (E) (numbering according to Kabat EUindex);and wherein furtherthe second antigen binding domain under b) is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst antigen binding domain under a), and the first antigen bindingdomain under a) and the third antigen binding domain under b) are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain under c).

In one aspect according to these aspects of the invention, in the firstsubunit of the Fc domain the threonine residue at position 366 isreplaced with a tryptophan residue (T366W), and in the second subunit ofthe Fc domain the tyrosine residue at position 407 is replaced with avaline residue (Y407V) and optionally the threonine residue at position366 is replaced with a serine residue (T366S) and the leucine residue atposition 368 is replaced with an alanine residue (L368A) (numberingsaccording to Kabat EU index).

In a further aspect according to these aspects of the invention, in thefirst subunit of the Fc domain additionally the serine residue atposition 354 is replaced with a cysteine residue (S354C) or the glutamicacid residue at position 356 is replaced with a cysteine residue (E356C)(particularly the serine residue at position 354 is replaced with acysteine residue), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C) (numberings according to Kabat EU index).

In still a further aspect according to these aspects of the invention,in each of the first and the second subunit of the Fc domain the leucineresidue at position 234 is replaced with an alanine residue (L234A), theleucine residue at position 235 is replaced with an alanine residue(L235A) and the proline residue at position 329 is replaced by a glycineresidue (P329G) (numbering according to Kabat EU index).

In still a further aspect according to these aspects of the invention,the Fc domain is a human IgG₁ Fc domain.

In a preferred specific aspect, the (multispecific) antibody comprises apolypeptide comprising an amino acid sequence that is at least 95%, 96%,97%, 98%, or 99% identical to the sequence of SEQ ID NO: 23 or SEQ IDNO: 39 (particularly SEQ ID NO: 39), a polypeptide comprising an aminoacid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 24, a polypeptide (particularly twopolypeptides) comprising an amino acid sequence that is at least 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 25, and apolypeptide comprising an amino acid sequence that is at least 95%, 96%,97%, 98%, or 99% identical to the sequence of SEQ ID NO: 27. In afurther preferred specific aspect, the (multispecific) antibodycomprises a polypeptide comprising the amino acid sequence of SEQ ID NO:23 or SEQ ID NO: 39 (particularly SEQ ID NO: 39), a polypeptidecomprising the amino acid sequence of SEQ ID NO: 24, a polypeptide(particularly two polypeptides)comprising the amino acid sequence of SEQID NO: 25 and a polypeptide comprising the amino acid sequence of SEQ IDNO: 27.

In one aspect the invention provides a (multispecific) antibody thatbinds to CD3 and CD19, comprising a polypeptide comprising an amino acidsequence that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 23 or SEQ ID NO: 39 (particularly SEQ ID NO: 39),a polypeptide comprising an amino acid sequence that is at least 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 24, apolypeptide (particularly two polypeptides)comprising an amino acidsequence that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 25, and a polypeptide comprising an amino acidsequence that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 27. In one aspect the invention provides a(multispecific) antibody that binds to CD3 and CD19, comprising apolypeptide comprising the amino acid sequence of SEQ ID NO: 23 or SEQID NO: 39 (particularly SEQ ID NO: 39), a polypeptide comprising theamino acid sequence of SEQ ID NO: 24, a polypeptide (particularly twopolypeptides) comprising the amino acid sequence of SEQ ID NO: 25 and apolypeptide comprising the amino acid sequence of SEQ ID NO: 27.

In a preferred specific aspect, the (multispecific) antibody comprises apolypeptide comprising an amino acid sequence that is at least 95%, 96%,97%, 98%, or 99% identical to the sequence of SEQ ID NO: 36 or SEQ IDNO: 40 (particularly SEQ ID NO: 36), a polypeptide comprising an aminoacid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 37, a polypeptide (particularly twopolypeptides) comprising an amino acid sequence that is at least 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 38, and apolypeptide comprising an amino acid sequence that is at least 95%, 96%,97%, 98%, or 99% identical to the sequence of SEQ ID NO: 27. In afurther preferred specific aspect, the (multispecific) antibodycomprises a polypeptide comprising the amino acid sequence of SEQ ID NO:36 or SEQ ID NO: 40 (particularly SEQ ID NO: 36), a polypeptidecomprising the amino acid sequence of SEQ ID NO: 37, a polypeptide(particularly two polypeptides) comprising the amino acid sequence ofSEQ ID NO: 38 and a polypeptide comprising the amino acid sequence ofSEQ ID NO: 27.

In one aspect the invention provides a (multispecific) antibody thatbinds to CD3 and CD19, comprising a polypeptide comprising an amino acidsequence that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 36 or SEQ ID NO: 40 (particularly SEQ ID NO: 36),a polypeptide comprising an amino acid sequence that is at least 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 37, apolypeptide (particularly two polypeptides) comprising an amino acidsequence that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 38, and a polypeptide comprising an amino acidsequence that is at least 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 27. In one aspect the invention provides a(multispecific) antibody that binds to CD3 and CD19, comprising apolypeptide comprising the amino acid sequence of SEQ ID NO: 36 or SEQID NO: 40 (particularly SEQ ID NO: 36), a polypeptide comprising theamino acid sequence of SEQ ID NO: 37, a polypeptide (particularly twopolypeptides) comprising the amino acid sequence of SEQ ID NO: 38 and apolypeptide comprising the amino acid sequence of SEQ ID NO: 27.

8. Fc Domain Variants

In preferred aspects, the (multispecific) antibody of the inventioncomprises an Fc domain composed of a first and a second subunit.

The Fc domain of the (multispecific) antibody consists of a pair ofpolypeptide chains comprising heavy chain domains of an immunoglobulinmolecule. For example, the Fc domain of an immunoglobulin G (IgG)molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgGheavy chain constant domains. The two subunits of the Fc domain arecapable of stable association with each other. In one aspect, the(multispecific) antibody of the invention comprises not more than one Fcdomain.

In one aspect, the Fc domain of the (multispecific) antibody is an IgGFc domain. In a preferred aspect, the Fc domain is an IgG₁ Fc domain. Inanother aspect the Fc domain is an IgG₄ Fc domain. In a more specificaspect, the Fc domain is an IgG₄ Fc domain comprising an amino acidsubstitution at position S228 (Kabat EU index numbering), particularlythe amino acid substitution S228P. This amino acid substitution reducesin vivo Fab arm exchange of IgG₄ antibodies (see Stubenrauch et al.,Drug Metabolism and Disposition 38, 84-91 (2010)). In a furtherpreferred aspect, the Fc domain is a human Fc domain. In an even morepreferred aspect, the Fc domain is a human IgG₁ Fc domain. An exemplarysequence of a human IgG₁ Fc region is given in SEQ ID NO: 47.

a) Fc Domain Modifications Promoting Heterodimerization

(Multispecific) antibodies according to the invention comprise differentantigen binding domains, which may be fused to one or the other of thetwo subunits of the Fc domain, thus the two subunits of the Fc domainare typically comprised in two non-identical polypeptide chains.Recombinant co-expression of these polypeptides and subsequentdimerization leads to several possible combinations of the twopolypeptides. To improve the yield and purity of (multispecific)antibodies in recombinant production, it will thus be advantageous tointroduce in the Fc domain of the (multispecific) antibody amodification promoting the association of the desired polypeptides.

Accordingly, in preferred aspects, the Fc domain of the (multispecific)antibody according to the invention comprises a modification promotingthe association of the first and the second subunit of the Fc domain.The site of most extensive protein-protein interaction between the twosubunits of a human IgG Fc domain is in the CH3 domain of the Fc domain.Thus, in one aspect said modification is in the CH3 domain of the Fcdomain.

There exist several approaches for modifications in the CH3 domain ofthe Fc domain in order to enforce heterodimerization, which are welldescribed e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205,WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, inall such approaches the CH3 domain of the first subunit of the Fc domainand the CH3 domain of the second subunit of the Fc domain are bothengineered in a complementary manner so that each CH3 domain (or theheavy chain comprising it) can no longer homodimerize with itself but isforced to heterodimerize with the complementarily engineered other CH3domain (so that the first and second CH3 domain heterodimerize and nohomdimers between the two first or the two second CH3 domains areformed). These different approaches for improved heavy chainheterodimerization are contemplated as different alternatives incombination with the heavy-light chain modifications (e.g. VH and VLexchange/replacement in one binding arm and the introduction ofsubstitutions of charged amino acids with opposite charges in the CH1/CLinterface) in the (multispecific) antibody which reduce heavy/lightchain mispairing and Bence Jones-type side products.

In a specific aspect said modification promoting the association of thefirst and the second subunit of the Fc domain is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in a preferred aspect, in the CH3 domain of the firstsubunit of the Fc domain of the (multispecific) antibody an amino acidresidue is replaced with an amino acid residue having a larger sidechain volume, thereby generating a protuberance within the CH3 domain ofthe first subunit which is positionable in a cavity within the CH3domain of the second subunit, and in the CH3 domain of the secondsubunit of the Fc domain an amino acid residue is replaced with an aminoacid residue having a smaller side chain volume, thereby generating acavity within the CH3 domain of the second subunit within which theprotuberance within the CH3 domain of the first subunit is positionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), and tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), and valine (V).

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis.

In a specific aspect, in (the CH3 domain of) the first subunit of the Fcdomain (the “knobs” subunit) the threonine residue at position 366 isreplaced with a tryptophan residue (T366W), and in (the CH3 domain of)the second subunit of the Fc domain (the “hole” subunit) the tyrosineresidue at position 407 is replaced with a valine residue (Y407V). Inone aspect, in the second subunit of the Fc domain additionally thethreonine residue at position 366 is replaced with a serine residue(T366S) and the leucine residue at position 368 is replaced with analanine residue (L368A) (numberings according to Kabat EU index).

In yet a further aspect, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C) or the glutamic acid residue at position 356 isreplaced with a cysteine residue (E356C) (particularly the serineresidue at position 354 is replaced with a cysteine residue), and in thesecond subunit of the Fc domain additionally the tyrosine residue atposition 349 is replaced by a cysteine residue (Y349C) (numberingsaccording to Kabat EU index). Introduction of these two cysteineresidues results in formation of a disulfide bridge between the twosubunits of the Fc domain, further stabilizing the dimer (Carter, JImmunol Methods 248, 7-15 (2001)).

In a preferred aspect, the first subunit of the Fc domain comprises theamino acid substitutions S354C and T366W, and the second subunit of theFc domain comprises the amino acid substitutions Y349C, T366S, L368A andY407V (numbering according to Kabat EU index). In a preferred aspect theantigen binding domain that binds to CD3 is fused (optionally via thesecond antigen binding domain, which binds to CD19, and/or a peptidelinker) to the first subunit of the Fc domain (comprising the “knob”modification). Without wishing to be bound by theory, fusion of theantigen binding domain that binds CD3 to the knob-containing subunit ofthe Fc domain will (further) minimize the generation of antibodiescomprising two antigen binding domains that bind to CD3 (steric clash oftwo knob-containing polypeptides).

Other techniques of CH3-modification for enforcing theheterodimerization are contemplated as alternatives according to theinvention and are described e.g. in WO 96/27011, WO 98/050431, EP1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304,WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO2013/096291.

In one aspect, the heterodimerization approach described in EP 1870459,is used alternatively. This approach is based on the introduction ofcharged amino acids with opposite charges at specific amino acidpositions in the CH3/CH3 domain interface between the two subunits ofthe Fc domain. A particular aspect for the (multispecific) antibody ofthe invention are amino acid mutations R409D; K370E in one of the twoCH3 domains (of the Fc domain) and amino acid mutations D399K; E357K inthe other one of the CH3 domains of the Fc domain (numbering accordingto Kabat EU index).

In another aspect, the (multispecific) antibody of the inventioncomprises amino acid mutation T366W in the CH3 domain of the firstsubunit of the Fc domain and amino acid mutations T366S, L368A, Y407V inthe CH3 domain of the second subunit of the Fc domain, and additionallyamino acid mutations R409D; K370E in the CH3 domain of the first subunitof the Fc domain and amino acid mutations D399K; E357K in the CH3 domainof the second subunit of the Fc domain (numberings according to Kabat EUindex).

In another aspect, the (multispecific) antibody of the inventioncomprises amino acid mutations S354C, T366W in the CH3 domain of thefirst subunit of the Fc domain and amino acid mutations Y349C, T366S,L368A, Y407V in the CH3 domain of the second subunit of the Fc domain,or said (multispecific) antibody comprises amino acid mutations Y349C,T366W in the CH3 domain of the first subunit of the Fc domain and aminoacid mutations S354C, T366S, L368A, Y407V in the CH3 domains of thesecond subunit of the Fc domain and additionally amino acid mutationsR409D; K370E in the CH3 domain of the first subunit of the Fc domain andamino acid mutations D399K; E357K in the CH3 domain of the secondsubunit of the Fc domain (all numberings according to Kabat EU index).

In one aspect, the heterodimerization approach described in WO2013/157953 is used alternatively. In one aspect, a first CH3 domaincomprises amino acid mutation T366K and a second CH3 domain comprisesamino acid mutation L351D (numberings according to Kabat EU index). In afurther aspect, the first CH3 domain comprises further amino acidmutation L351K.

In a further aspect, the second CH3 domain comprises further an aminoacid mutation selected from Y349E, Y349D and L368E (particularly L368E)(numberings according to Kabat EU index). In one aspect, theheterodimerization approach described in WO 2012/058768 is usedalternatively. In one aspect a first CH3 domain comprises amino acidmutations L351Y, Y407A and a second CH3 domain comprises amino acidmutations T366A, K409F. In a further aspect the second CH3 domaincomprises a further amino acid mutation at position T411, D399, 5400,F405, N390, or K392, e.g. selected from a) T411N, T411R, T411Q, T411K,T411D, T411E or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, 5400D,5400R, or 5400K, d) F4051, F405M, F405T, F4055, F405V or F405W, e)N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E(numberings according to Kabat EU index). In a further aspect a firstCH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3domain comprises amino acid mutations T366V, K409F. In a further aspect,a first CH3 domain comprises amino acid mutation Y407A and a second CH3domain comprises amino acid mutations T366A, K409F. In a further aspect,the second CH3 domain further comprises amino acid mutations K392E,T411E, D399R and 5400R (numberings according to Kabat EU index).

In one aspect, the heterodimerization approach described in WO2011/143545 is used alternatively, e.g. with the amino acid modificationat a position selected from the group consisting of 368 and 409(numbering according to Kabat EU index).

In one aspect, the heterodimerization approach described in WO2011/090762, which also uses the knobs-into-holes technology describedabove, is used alternatively. In one aspect a first CH3 domain comprisesamino acid mutation T366W and a second CH3 domain comprises amino acidmutation Y407A. In one aspect, a first CH3 domain comprises amino acidmutation T366Y and a second CH3 domain comprises amino acid mutationY407T (numberings according to Kabat EU index).

In one aspect, the (multispecific) antibody or its Fc domain is of IgG₂subclass and the heterodimerization approach described in WO 2010/129304is used alternatively.

In an alternative aspect, a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable. In one such aspect, a first CH3 domaincomprises amino acid substitution of K392 or N392 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D),particularly K392D or N392D) and a second CH3 domain comprises aminoacid substitution of D399, E356, D356, or E357 with a positively chargedamino acid (e.g. lysine (K) or arginine (R), particularly D399K, E356K,D356K, or E357K, and more particularly D399K and E356K). In a furtheraspect, the first CH3 domain further comprises amino acid substitutionof K409 or R409 with a negatively charged amino acid (e.g. glutamic acid(E), or aspartic acid (D), particularly K409D or R409D). In a furtheraspect the first CH3 domain further or alternatively comprises aminoacid substitution of K439 and/or K370 with a negatively charged aminoacid (e.g. glutamic acid (E), or aspartic acid (D)) (all numberingsaccording to Kabat EU index).

In yet a further aspect, the heterodimerization approach described in WO2007/147901 is used alternatively. In one aspect, a first CH3 domaincomprises amino acid mutations K253E, D282K, and K322D and a second CH3domain comprises amino acid mutations D239K, E240K, and K292D(numberings according to Kabat EU index).

In still another aspect, the heterodimerization approach described in WO2007/110205 can be used alternatively.

In one aspect, the first subunit of the Fc domain comprises amino acidsubstitutions K392D and K409D, and the second subunit of the Fc domaincomprises amino acid substitutions D356K and D399K (numbering accordingto Kabat EU index).

b) Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

The Fc domain confers to the (multispecific) antibody favorablepharmacokinetic properties, including a long serum half-life whichcontributes to good accumulation in the target tissue and a favorabletissue-blood distribution ratio. At the same time it may, however, leadto undesirable targeting of the (multispecific) antibody to cellsexpressing Fc receptors rather than to the preferred antigen-bearingcells. Moreover, the co-activation of Fc receptor signaling pathways maylead to cytokine release which, in combination with the T cellactivating properties and the long half-life of the (multispecific)antibody, results in excessive activation of cytokine receptors andsevere side effects upon systemic administration. Activation of (Fcreceptor-bearing) immune cells other than T cells may even reduceefficacy of the (multispecific) antibody due to the potentialdestruction of T cells e.g. by NK cells.

Accordingly, in preferred aspects, the Fc domain of the (multispecific)antibody according to the invention exhibits reduced binding affinity toan Fc receptor and/or reduced effector function, as compared to a nativeIgG₁ Fc domain. In one such aspect the Fc domain (or the (multispecific)antibody comprising said Fc domain) exhibits less than 50%, particularlyless than 20%, more particularly less than 10% and most particularlyless than 5% of the binding affinity to an Fc receptor, as compared to anative IgG₁ Fc domain (or a (multispecific) antibody comprising a nativeIgG₁ Fc domain), and/or less than 50%, particularly less than 20%, moreparticularly less than 10% and most particularly less than 5% of theeffector function, as compared to a native IgG₁ Fc domain domain (or a(multispecific) antibody comprising a native IgG₁ Fc domain). In oneaspect, the Fc domain domain (or the (multispecific) antibody comprisingsaid Fc domain) does not substantially bind to an Fc receptor and/orinduce effector function. In a preferred aspect the Fc receptor is anFcγ receptor. In one aspect the Fc receptor is a human Fc receptor. Inone aspect the Fc receptor is an activating Fc receptor. In a specificaspect the Fc receptor is an activating human Fcγ receptor, morespecifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically humanFcγRIIIa. In one aspect the effector function is one or more selectedfrom the group of CDC, ADCC, ADCP, and cytokine secretion. In apreferred aspect, the effector function is ADCC. In one aspect, the Fcdomain domain exhibits substantially similar binding affinity toneonatal Fc receptor (FcRn), as compared to a native IgG₁ Fc domaindomain. Substantially similar binding to FcRn is achieved when the Fcdomain (or the (multispecific) antibody comprising said Fc domain)exhibits greater than about 70%, particularly greater than about 80%,more particularly greater than about 90% of the binding affinity of anative IgG₁ Fc domain (or the (multispecific) antibody comprising anative IgG₁ Fc domain) to FcRn.

In certain aspects the Fc domain is engineered to have reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a non-engineered Fc domain. In preferred aspects, the Fc domain ofthe (multispecific) antibody comprises one or more amino acid mutationthat reduces the binding affinity of the Fc domain to an Fc receptorand/or effector function. Typically, the same one or more amino acidmutation is present in each of the two subunits of the Fc domain. In oneaspect, the amino acid mutation reduces the binding affinity of the Fcdomain to an Fc receptor. In one aspect, the amino acid mutation reducesthe binding affinity of the Fc domain to an Fc receptor by at least2-fold, at least 5-fold, or at least 10-fold. In aspects where there ismore than one amino acid mutation that reduces the binding affinity ofthe Fc domain to the Fc receptor, the combination of these amino acidmutations may reduce the binding affinity of the Fc domain to an Fcreceptor by at least 10-fold, at least 20-fold, or even at least50-fold. In one aspect the (multispecific) antibody comprising anengineered Fc domain exhibits less than 20%, particularly less than 10%,more particularly less than 5% of the binding affinity to an Fc receptoras compared to a (multispecific) antibody comprising a non-engineered Fcdomain. In a preferred aspect, the Fc receptor is an Fcγ receptor. Insome aspects, the Fc receptor is a human Fc receptor. In some aspects,the Fc receptor is an activating Fc receptor. In a specific aspect, theFc receptor is an activating human Fcγ receptor, more specifically humanFcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa.Preferably, binding to each of these receptors is reduced. In someaspects, binding affinity to a complement component, specificallybinding affinity to C1q, is also reduced. In one aspect, bindingaffinity to neonatal Fc receptor (FcRn) is not reduced. Substantiallysimilar binding to FcRn, i.e. preservation of the binding affinity ofthe Fc domain to said receptor, is achieved when the Fc domain (or the(multispecific) antibody comprising said Fc domain) exhibits greaterthan about 70% of the binding affinity of a non-engineered form of theFc domain (or the (multispecific) antibody comprising saidnon-engineered form of the Fc domain) to FcRn. The Fc domain, or(multispecific) antibodies of the invention comprising said Fc domain,may exhibit greater than about 80% and even greater than about 90% ofsuch affinity. In certain aspects, the Fc domain of the (multispecific)antibody is engineered to have reduced effector function, as compared toa non-engineered Fc domain. The reduced effector function can include,but is not limited to, one or more of the following: reduced complementdependent cytotoxicity (CDC), reduced antibody-dependent cell-mediatedcytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis(ADCP), reduced cytokine secretion, reduced immune complex-mediatedantigen uptake by antigen-presenting cells, reduced binding to NK cells,reduced binding to macrophages, reduced binding to monocytes, reducedbinding to polymorphonuclear cells, reduced direct signaling inducingapoptosis, reduced crosslinking of target-bound antibodies, reduceddendritic cell maturation, or reduced T cell priming. In one aspect, thereduced effector function is one or more selected from the group ofreduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion.In a preferred aspect, the reduced effector function is reduced ADCC. Inone aspect the reduced ADCC is less than 20% of the ADCC induced by anon-engineered Fc domain (or a (multispecific) antibody comprising anon-engineered Fc domain). In one aspect, the amino acid mutation thatreduces the binding affinity of the Fc domain to an Fc receptor and/oreffector function is an amino acid substitution. In one aspect, the Fcdomain comprises an amino acid substitution at a position selected fromthe group of E233, L234, L235, N297, P331 and P329 (numberings accordingto Kabat EU index). In a more specific aspect, the Fc domain comprisesan amino acid substitution at a position selected from the group ofL234, L235 and P329 (numberings according to Kabat EU index). In someaspects, the Fc domain comprises the amino acid substitutions L234A andL235A (numberings according to Kabat EU index). In one such aspect, theFc domain is an IgG₁ Fc domain, particularly a human IgG₁ Fc domain. Inone aspect, the Fc domain comprises an amino acid substitution atposition P329. In a more specific aspect, the amino acid substitution isP329A or P329G, particularly P329G (numberings according to Kabat EUindex). In one aspect, the Fc domain comprises an amino acidsubstitution at position P329 and a further amino acid substitution at aposition selected from E233, L234, L235, N297 and P331 (numberingsaccording to Kabat EU index). In a more specific aspect, the furtheramino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D orP331S. In preferred aspects, the Fc domain comprises amino acidsubstitutions at positions P329, L234 and L235 (numberings according toKabat EU index). In more preferred aspects, the Fc domain comprises theamino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or“LALAPG”). Specifically, in preferred aspects, each subunit of the Fcdomain comprises the amino acid substitutions L234A, L235A and P329G(Kabat EU index numbering), i.e. in each of the first and the secondsubunit of the Fc domain the leucine residue at position 234 is replacedwith an alanine residue (L234A), the leucine residue at position 235 isreplaced with an alanine residue (L235A) and the proline residue atposition 329 is replaced by a glycine residue (P329G) (numberingaccording to Kabat EU index).

In one such aspect, the Fc domain is an IgG₁ Fc domain, particularly ahuman IgG₁ Fc domain. The “P329G LALA” combination of amino acidsubstitutions almost completely abolishes Fcγ receptor (as well ascomplement) binding of a human IgG₁ Fc domain, as described in PCTpublication no. WO 2012/130831, which is incorporated herein byreference in its entirety. WO 2012/130831 also describes methods ofpreparing such mutant Fc domains and methods for determining itsproperties such as Fc receptor binding or effector functions.

IgG₄ antibodies exhibit reduced binding affinity to Fc receptors andreduced effector functions as compared to IgG₁ antibodies. Hence, insome aspects, the Fc domain of the (multispecific) antibodies of theinvention is an IgG₄ Fc domain, particularly a human IgG₄ Fc domain. Inone aspect, the IgG₄ Fc domain comprises an amino acid substitution atposition S228, specifically the amino acid substitution S228P(numberings according to Kabat EU index). To further reduce its bindingaffinity to an Fc receptor and/or its effector function, in one aspect,the IgG₄ Fc domain comprises an amino acid substitution at positionL235, specifically the amino acid substitution L235E (numberingsaccording to Kabat EU index). In another aspect, the IgG₄ Fc domaincomprises an amino acid substitution at position P329, specifically theamino acid substitution P329G (numberings according to Kabat EU index).In a preferred aspect, the IgG₄ Fc domain comprises amino acidsubstitutions at positions S228, L235 and P329, specifically amino acidsubstitutions S228P, L235E and P329G (numberings according to Kabat EUindex). Such IgG₄ Fc domain mutants and their Fcγ receptor bindingproperties are described in PCT publication no. WO 2012/130831,incorporated herein by reference in its entirety.

In a preferred aspect, the Fc domain exhibiting reduced binding affinityto an Fc receptor and/or reduced effector function, as compared to anative IgG₁ Fc domain, is a human IgG₁ Fc domain comprising the aminoacid substitutions L234A, L235A and optionally P329G, or a human IgG₄ Fcdomain comprising the amino acid substitutions S228P, L235E andoptionally P329G (numberings according to Kabat EU index).

In certain aspects, N-glycosylation of the Fc domain has beeneliminated. In one such aspect, the Fc domain comprises an amino acidmutation at position N297, particularly an amino acid substitutionreplacing asparagine by alanine (N297A) or aspartic acid (N297D)(numberings according to Kabat EU index).

In addition to the Fc domains described hereinabove and in PCTpublication no. WO 2012/130831, Fc domains with reduced Fc receptorbinding and/or effector function also include those with substitution ofone or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329(U.S. Pat. No. 6,737,056) (numberings according to Kabat EU index). SuchFc mutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. No. 7,332,581).

Mutant Fc domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. Alternatively, binding affinity ofFc domains or (multispecific) antibodies comprising an Fc domain for Fcreceptors may be evaluated using cell lines known to express particularFc receptors, such as human NK cells expressing FcγIIIa receptor.

Effector function of an Fc domain, or a (multispecific) antibodycomprising an Fc domain, can be measured by methods known in the art.Examples of in vitro assays to assess ADCC activity of a molecule ofinterest are described in U.S. Pat. No. 5,500,362; Hellstrom et al. ProcNatl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc NatlAcad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemannet al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactiveassays may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, Calif.); and CytoTox 96® non-radioactive cytotoxicity assay(Promega, Madison, Wis.)). Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g. in a animal model such as thatdisclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

In some aspects, binding of the Fc domain to a complement component,specifically to C1q, is reduced. Accordingly, in some aspects whereinthe Fc domain is engineered to have reduced effector function, saidreduced effector function includes reduced CDC. C1q binding assays maybe carried out to determine whether the Fc domain, or the(multispecific) antibody comprising the Fc domain, is able to bind C1qand hence has CDC activity. See e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., JImmunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052(2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)). FcRnbinding and in vivo clearance/half life determinations can also beperformed using methods known in the art (see, e.g., Petkova, S. B. etal., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013/120929).

B. Polynucleotides

The invention further provides an isolated polynucleotide encoding anantibody of the invention. Said isolated polynucleotide may be a singlepolynucleotide or a plurality of polynucleotides. The polynucleotidesencoding (multispecific) antibodies of the invention may be expressed asa single polynucleotide that encodes the entire antibody or as multiple(e.g., two or more) polynucleotides that are co-expressed. Polypeptidesencoded by polynucleotides that are co-expressed may associate through,e.g., disulfide bonds or other means to form a functional antibody. Forexample, the light chain portion of an antibody may be encoded by aseparate polynucleotide from the portion of the antibody comprising theheavy chain of the antibody. When co-expressed, the heavy chainpolypeptides will associate with the light chain polypeptides to formthe antibody. In another example, the portion of the antibody comprisingone of the two Fc domain subunits and optionally (part of) one or moreFab molecules could be encoded by a separate polynucleotide from theportion of the antibody comprising the other of the two Fc domainsubunits and optionally (part of) a Fab molecule. When co-expressed, theFc domain subunits will associate to form the Fc domain.

In some aspects, the isolated polynucleotide encodes the entire antibodymolecule according to the invention as described herein. In otheraspects, the isolated polynucleotide encodes a polypeptide comprised inthe antibody according to the invention as described herein.

In certain aspects the polynucleotide or nucleic acid is DNA. In otheraspects, a polynucleotide of the present invention is RNA, for example,in the form of messenger RNA (mRNA). RNA of the present invention may besingle stranded or double stranded.

C. Recombinant Methods

Antibodies of the invention may be obtained, for example, by solid-statepeptide synthesis (e.g. Merrifield solid phase synthesis) or recombinantproduction. For recombinant production one or more polynucleotideencoding the antibody, e.g., as described above, is isolated andinserted into one or more vectors for further cloning and/or expressionin a host cell. Such polynucleotide may be readily isolated andsequenced using conventional procedures. In one aspect a vector,particularly an expression vector, comprising the polynucleotide (i.a. asingle polynucleotide or a plurality of polynucleotides) of theinvention is provided. Methods which are well known to those skilled inthe art can be used to construct expression vectors containing thecoding sequence of an antibody along with appropriatetranscriptional/translational control signals. These methods include invitro recombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. See, for example, the techniquesdescribed in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL,Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and WileyInterscience, N.Y (1989). The expression vector can be part of aplasmid, virus, or may be a nucleic acid fragment. The expression vectorincludes an expression cassette into which the polynucleotide encodingthe antibody (i.e. the coding region) is cloned in operable associationwith a promoter and/or other transcription or translation controlelements. As used herein, a “coding region” is a portion of nucleic acidwhich consists of codons translated into amino acids. Although a “stopcodon” (TAG, TGA, or TAA) is not translated into an amino acid, it maybe considered to be part of a coding region, if present, but anyflanking sequences, for example promoters, ribosome binding sites,transcriptional terminators, introns, 5′ and 3′ untranslated regions,and the like, are not part of a coding region. Two or more codingregions can be present in a single polynucleotide construct, e.g. on asingle vector, or in separate polynucleotide constructs, e.g. onseparate (different) vectors. Furthermore, any vector may contain asingle coding region, or may comprise two or more coding regions, e.g. avector of the present invention may encode one or more polypeptides,which are post- or co-translationally separated into the final proteinsvia proteolytic cleavage. In addition, a vector, polynucleotide, ornucleic acid of the invention may encode heterologous coding regions,either fused or unfused to a polynucleotide encoding the antibody of theinvention, or variant or derivative thereof. Heterologous coding regionsinclude without limitation specialized elements or motifs, such as asecretory signal peptide or a heterologous functional domain. Anoperable association is when a coding region for a gene product, e.g. apolypeptide, is associated with one or more regulatory sequences in sucha way as to place expression of the gene product under the influence orcontrol of the regulatory sequence(s). Two DNA fragments (such as apolypeptide coding region and a promoter associated therewith) are“operably associated” if induction of promoter function results in thetranscription of mRNA encoding the desired gene product and if thenature of the linkage between the two DNA fragments does not interferewith the ability of the expression regulatory sequences to direct theexpression of the gene product or interfere with the ability of the DNAtemplate to be transcribed. Thus, a promoter region would be operablyassociated with a nucleic acid encoding a polypeptide if the promoterwas capable of effecting transcription of that nucleic acid. Thepromoter may be a cell-specific promoter that directs substantialtranscription of the DNA only in predetermined cells. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptioncontrol regions are disclosed herein. A variety of transcription controlregions are known to those skilled in the art. These include, withoutlimitation, transcription control regions, which function in vertebratecells, such as, but not limited to, promoter and enhancer segments fromcytomegaloviruses (e.g. the immediate early promoter, in conjunctionwith intron-A), simian virus 40 (e.g. the early promoter), andretroviruses (such as, e.g. Rous sarcoma virus). Other transcriptioncontrol regions include those derived from vertebrate genes such asactin, heat shock protein, bovine growth hormone and rabbit β-globin, aswell as other sequences capable of controlling gene expression ineukaryotic cells. Additional suitable transcription control regionsinclude tissue-specific promoters and enhancers as well as induciblepromoters (e.g. promoters inducible by tetracyclins). Similarly, avariety of translation control elements are known to those of ordinaryskill in the art. These include, but are not limited to ribosome bindingsites, translation initiation and termination codons, and elementsderived from viral systems (particularly an internal ribosome entrysite, or IRES, also referred to as a CITE sequence). The expressioncassette may also include other features such as an origin ofreplication, and/or chromosome integration elements such as retrovirallong terminal repeats (LTRs), or adeno-associated viral (AAV) invertedterminal repeats (ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the antibody is desired, DNA encoding a signal sequence may be placedupstream of the nucleic acid encoding an antibody of the invention or afragment thereof. According to the signal hypothesis, proteins secretedby mammalian cells have a signal peptide or secretory leader sequencewhich is cleaved from the mature protein once export of the growingprotein chain across the rough endoplasmic reticulum has been initiated.Those of ordinary skill in the art are aware that polypeptides secretedby vertebrate cells generally have a signal peptide fused to theN-terminus of the polypeptide, which is cleaved from the translatedpolypeptide to produce a secreted or “mature” form of the polypeptide.In certain aspects, the native signal peptide, e.g. an immunoglobulinheavy chain or light chain signal peptide is used, or a functionalderivative of that sequence that retains the ability to direct thesecretion of the polypeptide that is operably associated with it.Alternatively, a heterologous mammalian signal peptide, or a functionalderivative thereof, may be used. For example, the wild-type leadersequence may be substituted with the leader sequence of human tissueplasminogen activator (TPA) or mouse β-glucuronidase. DNA encoding ashort protein sequence that could be used to facilitate laterpurification (e.g. a histidine tag) or assist in labeling the antibodymay be included within or at the ends of the antibody (fragment)encoding polynucleotide.

In a further aspect, a host cell comprising a polynucleotide (i.e. asingle polynucleotide or a plurality of polynucleotides) of theinvention is provided. In certain aspects a host cell comprising avector of the invention is provided. The polynucleotides and vectors mayincorporate any of the features, singly or in combination, describedherein in relation to polynucleotides and vectors, respectively. In onesuch aspect a host cell comprises (e.g. has been transformed ortransfected with) one or more vector comprising one or morepolynucleotide that encodes (part of) an antibody of the invention. Asused herein, the term “host cell” refers to any kind of cellular systemwhich can be engineered to generate the antibody of the invention orfragments thereof. Host cells suitable for replicating and forsupporting expression of antibodies are well known in the art. Suchcells may be transfected or transduced as appropriate with theparticular expression vector and large quantities of vector containingcells can be grown for seeding large scale fermenters to obtainsufficient quantities of the antibody for clinical applications.Suitable host cells include prokaryotic microorganisms, such as E. coli,or various eukaryotic cells, such as Chinese hamster ovary cells (CHO),insect cells, or the like. For example, polypeptides may be produced inbacteria in particular when glycosylation is not needed. Afterexpression, the polypeptide may be isolated from the bacterial cellpaste in a soluble fraction and can be further purified. In addition toprokaryotes, eukaryotic microbes such as filamentous fungi or yeast aresuitable cloning or expression hosts for polypeptide-encoding vectors,including fungi and yeast strains whose glycosylation pathways have been“humanized”, resulting in the production of a polypeptide with apartially or fully human glycosylation pattern. See Gemgross, NatBiotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215(2006). Suitable host cells for the expression of (glycosylated)polypeptides are also derived from multicellular organisms(invertebrates and vertebrates). Examples of invertebrate cells includeplant and insect cells. Numerous baculoviral strains have beenidentified which may be used in conjunction with insect cells,particularly for transfection of Spodoptera frugiperda cells. Plant cellcultures can also be utilized as hosts. See e.g. U.S. Pat. Nos.5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describingPLANTIBODIES™ technology for producing antibodies in transgenic plants).Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293Tcells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)),baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkeykidney cells (CV1), African green monkey kidney cells (VERO-76), humancervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo ratliver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT 060562), TRI cells (as described,e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5cells, and FS4 cells. Other useful mammalian host cell lines includeChinese hamster ovary (CHO) cells, including dhfr⁻ CHO cells (Urlaub etal., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell linessuch as YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian hostcell lines suitable for protein production, see, e.g., Yazaki and Wu,Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,Totowa, N.J.), pp. 255-268 (2003). Host cells include cultured cells,e.g., mammalian cultured cells, yeast cells, insect cells, bacterialcells and plant cells, to name only a few, but also cells comprisedwithin a transgenic animal, transgenic plant or cultured plant or animaltissue. In one aspect, the host cell is a eukaryotic cell, particularlya mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a humanembryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20cell). In one aspect, the host cell is not a cell within a human body.Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a polypeptide comprising either theheavy or the light chain of an antigen binding domain such as anantibody, may be engineered so as to also express the other of theantibody chains such that the expressed product is an antibody that hasboth a heavy and a light chain. In one aspect, a method of producing anantibody according to the invention is provided, wherein the methodcomprises culturing a host cell comprising a polynucleotide encoding theantibody, as provided herein, under conditions suitable for expressionof the antibody, and optionally recovering the antibody from the hostcell (or host cell culture medium).

The components of the (multispecific) antibody of the invention may begenetically fused to each other. The (multispecific) antibody can bedesigned such that its components are fused directly to each other orindirectly through a linker sequence. The composition and length of thelinker may be determined in accordance with methods well known in theart and may be tested for efficacy. Examples of linker sequences betweendifferent components of (multispecific) antibodies are provided herein.Additional sequences may also be included to incorporate a cleavage siteto separate the individual components of the fusion if desired, forexample an endopeptidase recognition sequence.

Antibodies prepared as described herein may be purified by art-knowntechniques such as high performance liquid chromatography, ion exchangechromatography, gel electrophoresis, affinity chromatography, sizeexclusion chromatography, and the like. The actual conditions used topurify a particular protein will depend, in part, on factors such as netcharge, hydrophobicity, hydrophilicity etc., and will be apparent tothose having skill in the art. For affinity chromatography purification,an antibody, ligand, receptor or antigen can be used to which theantibody binds. For example, for affinity chromatography purification ofantibodies of the invention, a matrix with protein A or protein G may beused. Sequential Protein A or G affinity chromatography and sizeexclusion chromatography can be used to isolate an antibody essentiallyas described in the Examples. The purity of the antibody can bedetermined by any of a variety of well-known analytical methodsincluding gel electrophoresis, high pressure liquid chromatography, andthe like.

D. Assays

Antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

1. Binding Assays

The binding (affinity) of the antibody to an Fc receptor or a targetantigen can be determined for example by surface plasmon resonance(SPR), using standard instrumentation such as a BIAcore instrument (GEHealthcare), and receptors or target proteins such as may be obtained byrecombinant expression. Alternatively, binding of antibodies todifferent receptors or target antigens may be evaluated using cell linesexpressing the particular receptor or target antigen, for example byflow cytometry (FACS). A specific illustrative and exemplary aspect formeasuring binding activity to CD3 is described in the following.

In one aspect, the binding activity to CD3 is determined by SPR asfollows:

SPR is performed on a Biacore T200 instrument (GE Healthcare). Anti-Fabcapturing antibody (GE Healthcare, #28958325) is immobilized on a SeriesS Sensor Chip CMS (GE Healthcare) using standard amine couplingchemistry, at a surface density of 4000-6000 resonance units (RU). Asrunning and dilution buffer, HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4,0.05% Surfactant P20) is used. CD3 antibodies with a concentration of 2μg/ml (in 20 mM His, 140 mM NaCl, pH 6.0) are injected for about 60 s ata flow rate of 5 μl/min. The CD3 antigen used is a heterodimer of CD3delta and CD3 epsilon ectodomains fused to a human Fc domain withknob-into-hole modifications and a C-terminal Avi-tag (see SEQ ID NOs 41and 42). CD3 antigen is injected at a concentration of 10 μg/ml for 120s and dissociation is monitored at a flow rate of 5 μl/min for about 120s. The chip surface is regenerated by two consecutive injections of 10mM glycine pH 2.1 for about 60 s each. Bulk refractive index differencesare corrected by subtracting blank injections and by subtracting theresponse obtained from the blank control flow cell. For evaluation, thebinding response is taken 5 seconds after injection end. To normalizethe binding signal, the CD3 binding is divided by the anti-Fab response(the signal (RU) obtained upon capture of the CD3 antibody on theimmobilized anti-Fab antibody). The binding activity to CD3 of anantibody after a certain treatment, relative to the binding activity toCD3 of the antibody after a different treatment (also referred to asrelative active concentration (RAC)) is calculated by referencing thebinding activity of a sample of the antibody after the certain treatmentto the binding activity of a corresponding sample of the antibody afterthe different treatment.

2. Activity Assays

Biological activity of the (multispecific) antibodies of the inventioncan be measured by various assays as described in the Examples.Biological activities may for example include the induction ofproliferation of T cells, the induction of signaling in T cells, theinduction of expression of activation markers in T cells, the inductionof cytokine secretion by T cells, the induction of lysis of target cellssuch as B-cells, and the induction of tumor regression and/or theimprovement of survival.

E. Compositions, Formulations, and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the antibodies provided herein, e.g., for use in anyof the below therapeutic methods. In one aspect, a pharmaceuticalcomposition comprises an antibody according to the invention and apharmaceutically acceptable carrier. In another aspect, a pharmaceuticalcomposition comprises an antibody according to the invention and atleast one additional therapeutic agent, e.g., as described below.

Further provided is a method of producing an antibody of the inventionin a form suitable for administration in vivo, the method comprising (a)obtaining an antibody according to the invention, and (b) formulatingthe antibody with at least one pharmaceutically acceptable carrier,whereby a preparation of antibody is formulated for administration invivo.

Pharmaceutical compositions of the present invention comprise aneffective amount of antibody dissolved or dispersed in apharmaceutically acceptable carrier. The phrase “pharmaceuticallyacceptable” refers to molecular entities and compositions that aregenerally non-toxic to recipients at the dosages and concentrationsemployed, i.e. do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains an antibody and optionally an additional active ingredient willbe known to those of skill in the art in light of the presentdisclosure, as exemplified by Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference.Moreover, for animal (e.g., human) administration, it will be understoodthat preparations should meet sterility, pyrogenicity, general safetyand purity standards as required by FDA Office of Biological Standardsor corresponding authorities in other countries. Preferred compositionsare lyophilized formulations or aqueous solutions. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,buffers, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g. antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, antioxidants,proteins, drugs, drug stabilizers, polymers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the pharmaceutical compositions is contemplated.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the antibodies of the invention may be formulated inaqueous solutions, particularly in physiologically compatible bufferssuch as Hanks' solution, Ringer's solution, or physiological salinebuffer. The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the antibodies maybe in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use. Sterile injectable solutions areprepared by incorporating the antibodies of the invention in therequired amount in the appropriate solvent with various of the otheringredients enumerated below, as required. Sterility may be readilyaccomplished, e.g., by filtration through sterile filtration membranes.Generally, dispersions are prepared by incorporating the varioussterilized active ingredients into a sterile vehicle which contains thebasic dispersion medium and/or the other ingredients. In the case ofsterile powders for the preparation of sterile injectable solutions,suspensions or emulsion, the preferred methods of preparation arevacuum-drying or freeze-drying techniques which yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered liquid medium thereof. The liquid mediumshould be suitably buffered if necessary and the liquid diluent firstrendered isotonic prior to injection with sufficient saline or glucose.The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less than 0.5 ng/mg protein. Suitable pharmaceuticallyacceptable carriers include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes. Activeingredients may be entrapped in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular aspects, prolonged absorption ofan injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions described previously, the antibodies mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, theantibodies may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Pharmaceutical compositions comprising the antibodies of the inventionmay be manufactured by means of conventional mixing, dissolving,emulsifying, encapsulating, entrapping or lyophilizing processes.Pharmaceutical compositions may be formulated in conventional mannerusing one or more physiologically acceptable carriers, diluents,excipients or auxiliaries which facilitate processing of the proteinsinto preparations that can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen.

The antibodies may be formulated into a composition in a free acid orbase, neutral or salt form. Pharmaceutically acceptable salts are saltsthat substantially retain the biological activity of the free acid orbase. These include the acid addition salts, e.g., those formed with thefree amino groups of a proteinaceous composition, or which are formedwith inorganic acids such as for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric or mandelicacid. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as for example, sodium, potassium, ammonium,calcium or ferric hydroxides; or such organic bases as isopropylamine,trimethylamine, histidine or procaine. Pharmaceutical salts tend to bemore soluble in aqueous and other protic solvents than are thecorresponding free base forms.

F. Therapeutic Methods and Compositions

Any of the antibodies provided herein may be used in therapeuticmethods. Antibodies of the invention may be used as immunotherapeuticagents, for example in the treatment of cancers or autoimmune diseases.

For use in therapeutic methods, antibodies of the invention would beformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners.

In one aspect, antibodies of the invention for use as a medicament areprovided. In further aspects, antibodies of the invention for use intreating a disease are provided. In certain aspects, antibodies of theinvention for use in a method of treatment are provided. In one aspect,the invention provides an antibody of the invention for use in thetreatment of a disease in an individual in need thereof. In certainaspects, the invention provides an antibody for use in a method oftreating an individual having a disease comprising administering to theindividual an effective amount of the antibody. In certain aspects thedisease is a proliferative disorder. In certain aspects the disease iscancer, particularly a CD19-expressing cancer. In a specific aspect thecancer is a B-cell cancer. The B-cell cancer in one aspect is a B-celllymphoma or a B-cell leukemia. In one aspect the B-cell cancer isnon-Hodgkin lymphoma or acute lymphoblastic leukemia or chroniclymphocytic leukemia. In other aspects the disease is an autoimmunedisease. In a specific aspect the disease is lupus, in particularsystemic lupus erythematosus (SLE) or lupus nephritis (LN).

In certain aspects the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, e.g., an anti-cancer agent if the disease to be treated is canceror an immunosuppressive agent if the the disease to be treated is anautoimmune disease. In further aspects, the invention provides anantibody of the invention for use in inducing lysis of a target cell,particularly a B-cell. In certain aspects, the invention provides anantibody of the invention for use in a method of inducing lysis of atarget cell, particularly a B-cell, in an individual comprisingadministering to the individual an effective amount of the antibody toinduce lysis of a target cell. An “individual” according to any of theabove aspects is a mammal, preferably a human.

In a further aspect, the invention provides for the use of an antibodyof the invention in the manufacture or preparation of a medicament. Inone aspect the medicament is for the treatment of a disease in anindividual in need thereof. In a further aspect, the medicament is foruse in a method of treating a disease comprising administering to anindividual having the disease an effective amount of the medicament. Incertain aspects the disease is a proliferative disorder. In certainaspects the disease is cancer, particularly a CD19-expressing cancer. Ina specific aspect the cancer is a B-cell cancer. The B-cell cancer inone aspect is a B-cell lymphoma or a B-cell leukemia. In one aspect theB-cell cancer is non-Hodgkin lymphoma or acute lymphoblastic leukemia orchronic lymphocytic leukemia. In other aspects the disease is anautoimmune disease. In a specific aspect the disease is lupus, inparticular systemic lupus erythematosus (SLE) or lupus nephritis (LN).In one aspect, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, e.g., an anti-cancer agent if the disease to be treated is canceror an immunosuppressive agent if the the disease to be treated is anautoimmune disease. In a further aspect, the medicament is for inducinglysis of a target cell, particularly a B-cell. In still a furtheraspect, the medicament is for use in a method of inducing lysis of atarget cell, particularly a B-cell, in an individual comprisingadministering to the individual an effective amount of the medicament toinduce lysis of a target cell. An “individual” according to any of theabove aspects may be a mammal, preferably a human.

In a further aspect, the invention provides a method for treating adisease. In one aspect, the method comprises administering to anindividual having such disease an effective amount of an antibody of theinvention. In one aspect a composition is administered to saidindividual, comprising the antibody of the invention in apharmaceutically acceptable form. In certain aspects the disease is aproliferative disorder. In certain aspects the disease is cancer,particularly a CD19-expressing cancer. In a specific aspect the canceris a B-cell cancer. The B-cell cancer in one aspect is a B-cell lymphomaor a B-cell leukemia. In one aspect the B-cell cancer is non-Hodgkinlymphoma or acute lymphoblastic leukemia or chronic lymphocyticleukemia. In other aspects the disease is an autoimmune disease. In aspecific aspect the disease is lupus, in particular systemic lupuserythematosus (SLE) or lupus nephritis (LN). In certain aspects themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, e.g., ananti-cancer agent if the disease to be treated is cancer or animmunosuppressive agent if the the disease to be treated is anautoimmune disease. An “individual” according to any of the aboveaspects may be a mammal, preferably a human.

In a further aspect, the invention provides a method for inducing lysisof a target cell, particularly a CD19-expressing cell such as a B-cell.In one aspect the method comprises contacting a target cell with anantibody of the invention in the presence of a T cell, particularly acytotoxic T cell. In a further aspect, a method for inducing lysis of atarget cell, particularly a CD19-expressing cell such as a B-cell, in anindividual is provided. In one such aspect, the method comprisesadministering to the individual an effective amount of an antibody ofthe invention to induce lysis of a target cell. In one aspect, an“individual” is a human.

A skilled artisan readily recognizes that in many cases the antibody maynot provide a cure but may only provide partial benefit. In someaspects, a physiological change having some benefit is also consideredtherapeutically beneficial. Thus, in some aspects, an amount of antibodythat provides a physiological change is considered an “effectiveamount”. The subject, patient, or individual in need of treatment istypically a mammal, more specifically a human.

In some aspects, an effective amount of an antibody of the invention isadministered to an individual for the treatment of disease.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the route of administration, the body weight ofthe patient, the type of antibody, the severity and course of thedisease, whether the antibody is administered for preventive ortherapeutic purposes, previous or concurrent therapeutic interventions,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The practitioner responsible foradministration will, in any event, determine the concentration of activeingredient(s) in a composition and appropriate dose(s) for theindividual subject. Various dosing schedules including but not limitedto single or multiple administrations over various time-points, bolusadministration, and pulse infusion are contemplated herein. The antibodyis suitably administered to the patient at one time or over a series oftreatments. Depending on the type and severity of the disease, about 1μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody can be aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. One typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs. One exemplary dosage of the antibody wouldbe in the range from about 0.005 mg/kg to about 10 mg/kg. In othernon-limiting examples, a dose may also comprise from about 1microgram/kg body weight, about 5 microgram/kg body weight, about 10microgram/kg body weight, about 50 microgram/kg body weight, about 100microgram/kg body weight, about 200 microgram/kg body weight, about 350microgram/kg body weight, about 500 microgram/kg body weight, about 1milligram/kg body weight, about 5 milligram/kg body weight, about 10milligram/kg body weight, about 50 milligram/kg body weight, about 100milligram/kg body weight, about 200 milligram/kg body weight, about 350milligram/kg body weight, about 500 milligram/kg body weight, to about1000 mg/kg body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg body weight to about100 mg/kg body weight, about 5 microgram/kg body weight to about 500milligram/kg body weight, etc., can be administered, based on thenumbers described above. Thus, one or more doses of about 0.5 mg/kg, 2.0mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may beadministered to the patient. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g. such that thepatient receives from about two to about twenty, or e.g. about six dosesof the antibody). An initial higher loading dose, followed by one ormore lower doses may be administered. However, other dosage regimens maybe useful. The progress of this therapy is easily monitored byconventional techniques and assays.

The antibodies of the invention will generally be used in an amounteffective to achieve the intended purpose. For use to treat or prevent adisease condition, the antibodies of the invention, or pharmaceuticalcompositions thereof, are administered or applied in an effectiveamount. For systemic administration, an effective dose can be estimatedinitially from in vitro assays, such as cell culture assays. A dose canthen be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art.

Dosage amount and interval may be adjusted individually to provideplasma levels of the antibodies which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 50 mg/kg/day, typically from about 0.5to 1 mg/kg/day. Therapeutically effective plasma levels may be achievedby administering multiple doses each day. Levels in plasma may bemeasured, for example, by HPLC.

An effective dose of the antibodies of the invention will generallyprovide therapeutic benefit without causing substantial toxicity.Toxicity and therapeutic efficacy of an antibody can be determined bystandard pharmaceutical procedures in cell culture or experimentalanimals. Cell culture assays and animal studies can be used to determinethe LD₅₀ (the dose lethal to 50% of a population) and the ED₅₀ (the dosetherapeutically effective in 50% of a population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, whichcan be expressed as the ratio LD₅₀/ED₅₀. Antibodies that exhibit largetherapeutic indices are preferred. In one aspect, the antibody accordingto the present invention exhibits a high therapeutic index. The dataobtained from cell culture assays and animal studies can be used informulating a range of dosages suitable for use in humans. The dosagelies preferably within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon a variety of factors, e.g., the dosage formemployed, the route of administration utilized, the condition of thesubject, and the like. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition (see, e.g., Fingl et al., 1975, in: ThePharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated hereinby reference in its entirety).

The attending physician for patients treated with antibodies of theinvention would know how and when to terminate, interrupt, or adjustadministration due to toxicity, organ dysfunction, and the like.Conversely, the attending physician would also know to adjust treatmentto higher levels if the clinical response were not adequate (precludingtoxicity). The magnitude of an administered dose in the management ofthe disorder of interest will vary with the severity of the condition tobe treated, with the route of administration, and the like. The severityof the condition may, for example, be evaluated, in part, by standardprognostic evaluation methods. Further, the dose and perhaps dosefrequency will also vary according to the age, body weight, and responseof the individual patient.

The antibodies of the invention may be administered in combination withone or more other agents in therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent. The term “therapeutic agent” encompasses any agentadministered to treat a symptom or disease in an individual in need ofsuch treatment. Such additional therapeutic agent may comprise anyactive ingredients suitable for the particular disease being treated,preferably those with complementary activities that do not adverselyaffect each other. In certain aspects, an additional therapeutic agentis an immunomodulatory agent, a cytostatic agent, an inhibitor of celladhesion, a cytotoxic agent, an activator of cell apoptosis, or an agentthat increases the sensitivity of cells to apoptotic inducers. Incertain aspects, the additional therapeutic agent is an anti-canceragent, for example a microtubule disruptor, an antimetabolite, atopoisomerase inhibitor, a DNA intercalator, an alkylating agent, ahormonal therapy, a kinase inhibitor, a receptor antagonist, anactivator of tumor cell apoptosis, or an antiangiogenic agent. In otheraspects, the additional therapeutic agent is an immunosuppressive agent.In a specific aspect, the additional therapeutic agent is one or moreselected from the group of corticosteroids, hydroxychloroquine,mycophenolate mofetil, mycophenolic acid, methotrexate, azathioprine,cyclophosphamide, calcineurin inhibitors, belimumab, rituximab andobinutuzumab.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of antibody used, the type ofdisorder or treatment, and other factors discussed above. The antibodiesare generally used in the same dosages and with administration routes asdescribed herein, or about from 1 to 99% of the dosages describedherein, or in any dosage and by any route that is empirically/clinicallydetermined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Antibodies of the invention may alsobe used in combination with radiation therapy.

G. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this aspect of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

H. Methods and Compositions for Diagnostics and Detection

In certain aspects, any of the antibodies provided herein is useful fordetecting the presence of its target (e.g. CD3 or CD19) in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain aspects, a biological sample comprisesa cell or tissue, such as prostate tissue.

In one aspect, an antibody according to the invention for use in amethod of diagnosis or detection is provided. In a further aspect, amethod of detecting the presence of CD3 or CD19 in a biological sampleis provided. In certain aspects, the method comprises contacting thebiological sample with an antibody of the present invention underconditions permissive for binding of the antibody to CD3 or CD19, anddetecting whether a complex is formed between the antibody and CD3 orCD19. Such method may be an in vitro or in vivo method. In one aspect,an antibody of the invention is used to select subjects eligible fortherapy with an antibody that binds CD3 and/or CD19, e.g. where CD3and/or CD19 is a biomarker for selection of patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include cancer, particularly B-cell cancer.

In certain aspects, an antibody according to the present invention isprovided, wherein the antibody is labelled. Labels include, but are notlimited to, labels or moieties that are detected directly (such asfluorescent, chromophoric, electron-dense, chemiluminescent, andradioactive labels), as well as moieties, such as enzymes or ligands,that are detected indirectly, e.g., through an enzymatic reaction ormolecular interaction. Exemplary labels include, but are not limited to,the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such asrare earth chelates or fluorescein and its derivatives, rhodamine andits derivatives, dansyl, umbelliferone, luceriferases, e.g., fireflyluciferase and bacterial luciferase (U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

III. Sequences

SEQ ID Amino Acid Sequence NO CD3_(orig) TYAMN 1 HCDR1 CD3_(opt) SYAMN 2HCDR1 CD3_(orig)/ RIRSKYNNYATYYADSVKG 3 CD3_(opt) HCDR2 CD3_(orig)HGNFGNSYVSWFAY 4 HCDR3 CD3_(opt) HTTFPSSYVSYYGY 5 HCDR3 CD3_(orig )VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQA 6PGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQG TLVTVSS CD3_(opt )VHEVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQA 7PGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYVVGQGT LVTVSS CD3_(orig)/GSSTGAVTTSNYAN 8 CD3_(opt) LCDR1 CD3_(orig)/ GTNKRAP 9 CD3_(opt) LCDR2CD3_(orig)/ ALWYSNLWV 10 CD3_(opt) LCDR3 CD3_(orig)/QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQE 11 CD3_(opt )VLKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQ PEDEAEYYCALWYSNLWVFGGGTKLTVLCD3_(orig )IgG EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQA 12 HCPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSP CD3_(opt )IgGEVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQA 13 HCPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYVVGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSP CD3_(orig)/QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQE 14 CD3_(opt )IgGKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQ LCPEDEAEYYCALWYSNLWVFGGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC CD19(2B11)DYIMH 15 HCDR1 CD19(2B11) YINPYNDGSKYTEKFQG 16 HCDR2 CD19(2B11)GTYYYGPQLFDY 17 HCDR3 CD19(2B11) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ18 VH APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVT VSS CD19(2B11) KSSQSLETSTGTTYLN19 LCDR1 CD19(2B11) RVSKRFS 20 LCDR2 CD19(2B11) LQLLEDPYT 21 LCDR3CD19(2B11) DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGTTYLNWYL 22 VLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCLQLLEDPYTFGQGTKLEIKCD19(2B11) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 23 VH-CH1(EE) -APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA CD3_(orig)/CD3_(opt)YMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVT VL-CH1 -VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTV Fc (knob,SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT PGLALA)YICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGGQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP CD19(2B11)QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 24 VH-CH1(EE)-APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA Fc (hole,YMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVT PGLALA)VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSP CD19(2B11)DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGTTYLNWYL 25 VL-CL(RK)QKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLLEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC CD3_(orig) VH-EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQA 26 CLPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC CD3_(opt )VH-EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQA 27 CLPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYVVGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGECCD19(018) DYIMH 28 HCDR1 CD19(018) YINPYNDGSKYTEKFQG 29 HCDR2 CD19(018)GTYYYGSALFDY 30 HCDR3 CD19(018) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ31 VH APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGSALFDYWGQGTTVT VSS CD19(018) KSSQSLENPNGNTYLN32 LCDR1 CD19(018) RVSKRFS 33 LCDR2 CD19(018) LQLTHVPYT 34 LCDR3CD19(018) DIVMTQTPLSLSVTPGQPASISCKSSQSLENPNGNTYLNWY 35 VLLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRV EAEDVGVYYCLQLTHVPYTFGQGTKLEIKCD19(018) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 36 VH-CH1(EE) -APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA CD3_(orig)/CD3_(opt)YMELSRLRSDDTAVYYCARGTYYYGSALFDYWGQGTTVT VL-CH1 -VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTV Fc (knob,SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT PGLALA)YICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGGQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP CD19(018)QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 37 VH-CH1(EE) -APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA Fc (hole,YMELSRLRSDDTAVYYCARGTYYYGSALFDYWGQGTTVT PGLALA)VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSP CD19(018)DIVMTQTPLSLSVTPGQPASISCKSSQSLENPNGNTYLNWY 38 VL-CL(RK)LQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLTHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC CD19(2B11)QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 39 VH-CH1(EE) -APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA CD3_(orig)/CD3_(opt)YMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVT VL-CH1 -VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTV Fc (knob,SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT PGLALA)YICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP CD19(018)QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 40 VH-CH1(EE) -APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA CD3_(orig)/CD3_(opt)YMELSRLRSDDTAVYYCARGTYYYGSALFDYWGQGTTVT VL-CH1 -VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTV Fc (knob,SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT PGLALAYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP Human CD3QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHN 41 epsilon stalk -DKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGS Fc(knob) -KPEDANFYLYLRARVSENCVDEQLYFQGGSPKSADKTHTC AviPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE Human CD3FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRI 42 delta stalk -LDPRGIYRCNGTDIYKDKESTVQVHYRMCRSEQLYFQGDK Fc (hole) -THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV AviDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE CD19 ECD -PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLK 43 Fc (knob) -PFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPG AviPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVDASGGSPTPPTPGGGSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGG LNDIFEAQKIEWHE Fc (hole)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV 44VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV FSCSVMHEALHNRFTQKSLSLSPHuman CD3 QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHN 45DKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYVVSKNRKAKAKPVTRGAGAGGRQRGQNKERPPP VPNPDYEPIRKGQRDLYSGLNQRRICynomolgus QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHN 46 CD3GKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPI RKGQQDLYSGLNQRRI hIgG₁ FcDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV 47 regionVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSP linkerGGGGSGGGGS 48 linker DGGGGSGGGGS 49 linker GGGGSGGGGG 50 linkerDGGGGSGGGGG 51 Human RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW 52kappa CL KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK domainHKVYACEVTHQGLSSPVTKSFNRGEC HumanQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW 53 lambda CLKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSH domain RSYSCQVTHEGSTVEKTVAPTECSHuman IgG₁ ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW 54 heavy chainNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC constantNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF region (CH1-LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG CH2-CH3)VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSP

IV. Examples

The following are examples of methods and compositions of the invention.It is understood that various other aspects may be practiced, given thegeneral description provided above.

Example 1—Generation of Optimized CD3 Binder

Starting from a previously described (see e.g. WO 2014/131712,incorporated herein by reference) CD3 binder, termed “CD3_(orig)” hereinand comprising the VH and VL sequences of SEQ ID NOs 6 and 11,respectively, we aimed at optimizing properties of this binder byremoval of two asparagine deamidation sequence motifs at Kabat positions97 and 100 of the heavy chain CDR3.

To this aim, we generated an antibody library, suitable for phagedisplay, of the heavy chain with both asparagines at Kabat position 97and 100 removed, and in addition the CDRs H1, H2, and H3 randomized inorder to compensate for loss of affinity caused by replacing Asn97 andAsn100 through an affinity-maturation process.

This library was put on a filamentous phage via fusion to minor coatprotein p3 (Marks et al. (1991) J Mol Biol 222, 581-597) and selectedfor binding to recombinant CD3ε.

10 candidate clones were identified in the initial screening, showingacceptable binding on recombinant antigen as measured by SPR as Fabfragments (produced in E. coli).

Only one of these clones, however, showed acceptable binding activity toCD3 expressing cells as measured by flow cytometry after conversion toIgG format.

The selected clone, termed “CD3_(opt)” herein and comprising the VH andVL sequences of SEQ ID NOs 7 and 11, respectively, was further evaluatedand converted into bispecific format as described in the following.

Example 2—Binding of Optimized CD3 Binder to CD3

Binding to Recombinant CD3

Binding to recombinant CD3 was determined by surface plasmon resonance(SPR) for the optimized CD3 binder “CD3_(opt)” and the original CD3binder “CD3_(orig)”, both in human IgG₁ format with P329G L234A L235A(“PGLALA”, EU numbering) mutations in the Fc region (SEQ ID NOs 12 and14 (CD3_(orig)) and SEQ ID NOs 13 and 14 (CD3_(opt))).

In order to assess the effect of the deamidation site removal and itseffect on the stability of the antibodies, binding of the original andthe optimized CD3 binder to recombinant CD3 was tested after temperaturestress for 14 days at 37° C. or 40° C. Samples stored at −80° C. wereused as reference. The reference samples and the samples stressed at 40°C. were in 20 mM His, 140 mM NaCl, pH 6.0, and the samples stressed at37° C. in PBS, pH 7.4, all at a concentration of 1.2-1.3 mg/ml. Afterthe stress period (14 days) samples in PBS were dialyzed back to 20 mMHis, 140 mM NaCl, pH 6.0 for further analysis.

Relative Active Concentration (RAC) of the samples was determined by SPRas follows.

SPR was performed on a Biacore T200 instrument (GE Healthcare). Anti-Fabcapturing antibody (GE Healthcare, #28958325) was immobilized on aSeries S Sensor Chip CMS (GE Healthcare) using standard amine couplingchemistry, resulting in a surface density of 4000-6000 resonance units(RU). As running and dilution buffer, HBS-P+ (10 mM HEPES, 150 mM NaClpH 7.4, 0.05% Surfactant P20) was used. CD3 antibodies with aconcentration of 2 μg/ml were injected for 60 s at a flow rate of 5 CD3antigen (see below) was injected at a concentration of 10 μg/ml for 120s and dissociation was monitored at a flow rate of 5 μl/min for 120 s.The chip surface was regenerated by two consecutive injections of 10 mMglycine pH 2.1 for 60 s each. Bulk refractive index differences werecorrected by subtracting blank injections and by subtracting theresponse obtained from the blank control flow cell. For evaluation, thebinding response was taken 5 seconds after injection end. To normalizethe binding signal, the CD3 binding was divided by the anti-Fab response(the signal (RU) obtained upon capture of the CD3 antibody on theimmobilized anti-Fab antibody). The relative active concentration wascalculated by referencing each temperature stressed sample to thecorresponding, non-stressed sample.

The antigen used was a heterodimer of CD3 delta and CD3 epsilonectodomains fused to a human Fc domain with knob-into-hole modificationsand a C-terminal Avi-tag (see SEQ ID NOs 41 and 42).

The results of this experiment are shown in FIG. 2. As can be seen, theoptimized CD3 binder CD3_(opt) showed strongly improved binding to CD3after temperature stress (2 weeks at 37° C., pH 7.4) as compared to theoriginal CD3 binder CD3_(orig). This result demonstrates that thedeamidation site removal was successful, and has yielded an antibodywith superior stability properties, relevant for in vivo half-life, aswell as formulation of the antibody at neutral pH.

Binding to CD3 on Jurkat Cells

Binding to CD3 on the human reporter T-cell line Jurkat NFAT wasdetermined by FACS for the optimized CD3 binder “CD3_(opt)” and theoriginal CD3 binder “CD3_(orig)”, both in human IgG₁ format with P329GL234A L235A (“PGLALA”, EU numbering) mutations in the Fc region (SEQ IDNOs 12 and 14 (CD3_(orig)) and SEQ ID NOs 13 and 14 (CD3_(opt))).

Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega#CS176501) are a human acute lymphatic leukemia reporter cell line witha NFAT promoter, expressing human CD3. The cells were cultured inRPMI1640, 2 g/l glucose, 2 g/l NaHCO₃, 10% FCS, 25 mM HEPES, 2 mML-glutamine, 1×NEAA, 1× sodium-pyruvate at 0.1-0.5 mio cells per ml. Afinal concentration of 200 μg per ml hygromycin B was added whenevercells were passaged.

For the binding assay, Jurkat NFAT cells were harvested, washed with PBSand resuspended in FACS buffer. The antibody staining was performed in a96-well round bottom plate. Therefore 100′000 to 200′000 cells wereseeded per well. The plate was centrifuged for 4 min at 400×g and thesupernatant was removed. The test antibodies were diluted in FACS bufferand 20 μl of the antibody solution were added to the cells for 30 min at4° C. To remove unbound antibody, the cells were washed twice with FACSbuffer before addition of the diluted secondary antibody (PE-conjugatedAffiniPure F(ab′)2 Fragment goat anti-human IgG Fcg Fragment Specific;Jackson ImmunoResearch #109-116-170). After 30 min incubation at 4° C.unbound secondary antibody was washed away. Before measurement the cellswere resuspended in 200 μl FACS buffer and then analyzed by flowcytometry using a BD Canto II device.

As shown in FIG. 3, the optimized CD3 binder “CD3_(opt)” and theoriginal CD3 binder “CD3_(orig)” bound comparably well to CD3 on Jurkatcells.

Example 3—Functional Activity of Optimized CD3 Binder

The functional activity of the optimized CD3 binder “CD3_(opt)” wastested in a Jurkat reporter cell assay and compared to the activity ofthe original CD3 binder “CD3_(orig)”. To test the functional activity ofthe IgGs, anti-PGLALA expressing CHO cells were co-incubated with JurkatNFAT reporter cells in the presence of increasing concentrations ofCD3_(opt) human IgG₁ PGLALA or CD3_(orig) human IgG₁ PGLALA. Activationof CD3 on the Jurkat NFAT reporter cells upon T cell cross-linkinginduces the production of luciferase and luminescence can be measured asan activation marker. CD3_(orig) human IgG₁ wt was included as negativecontrol which cannot bind to anti-PGLALA expressing CHO cells andtherefore cannot be crosslinked on Jurkat NFAT cells. A schematicillustration of the assay is provided in FIG. 4.

Anti-PGLALA expressing CHO cells are CHO-K1 cells engineered to expresson their surface an antibody that specifically binds human IgG₁Fc(PGLALA) (see WO 2017/072210, incorporated herein by reference). Thesecells were cultured in DMEM/F12 medium containing 5% FCS+1% GluMax. TheJurkat NFAT reporter cells are as described in Example 2.

Upon simultaneous binding of the CD3 huIgG₁ PGLALA to anti-PGLALAexpressed on CHO and CD3 expressed on Jurkat-NFAT reporter cells, theNFAT promoter is activated and leads to expression of active fireflyluciferase. The intensity of luminescence signal (obtained upon additionof luciferase substrate) is proportional to the intensity of CD3activation and signaling. Jurkat-NFAT reporter cells grow in suspensionand were cultured in RPMI1640, 2 g/l glucose, 2 g/l NaHCO₃, 10% FCS, 25mM HEPES, 2 mM L-glutamin, 1×NEAA, 1× sodium-pyruvate at 0.1-0.5 miocells per ml, 200 μg per ml hygromycin. For the assay, CHO cells wereharvested and viability determined using ViCell. 30 000 targetcells/well were plated in a flat-bottom, white-walled 96-well-plate(Greiner bio-one #655098) in 100 μl medium and 50 μl/well of dilutedantibodies or medium (for controls) were added to the CHO cells.Subsequently, Jurkat-NFAT reporter cells were harvested and viabilityassessed using ViCell. Cells were resuspended at 1.2 mio cells/ml incell culture medium without hygromycin B and added to CHO cells at 60000 cells/well (50 μl/well) to obtain a final effector-to-target (E:T)ratio of 2:1 and a final volume of 200 μl per well. Then, 4 μl ofGloSensor (Promega #E1291) was added to each well (2% of final volume).Cells were incubated for 24 h at 37° C. in a humidified incubator. Atthe end of incubation time, luminescence was detected using TECAN Spark10M.

As shown in FIG. 5, the optimized CD3 binder CD3_(opt) had a similaractivity on Jurkat NFAT cells upon crosslinking as CD3_(orig).

Example 4—Generation of T-Cell Bispecific Antibody Comprising OptimizedCD3 Binder

The optimized CD3 binder identified in Example 1 (“CD3_(opt)”, SEQ IDNOs 7 (VH) and 11 (VL)) was used to generate T-cell bispecificantibodies (TCBs) targeting CD3 and CD19 (“CD19-TCB”), using anti-CD19antibodies 2B11 or 018 as CD19 binding moiety (SEQ ID NOs 15-22 or28-35, respectively).

A schematic illustration of the TCB molecules is provided in FIG. 6A,and their full sequences are given in SEQ ID NOs 39, 24, 25 and 27(2B11), and SEQ ID NOs 36, 37, 38 and 27 (018).

Corresponding molecules comprising either of the above-mentionedanti-CD19 antibodies as target cell antigen binding moiety andCD3_(orig as CD)3 binder were also prepared (SEQ ID NOs 39, 24, 25 and26 (2B11), and SEQ ID NOs 40, 37, 38 and 26 (018)).

The variable region of heavy and light chain DNA sequences weresubcloned in frame with either the constant heavy chain or the constantlight chain pre-inserted into the respective recipient mammalianexpression vectors as shown in FIG. 6 B-E.

To improve correct pairing of the light chains with the correspondingheavy chains, mutations were introduced in the human CL (E123R, Q124K)and the human CH1 (K147E, K213E) of the CD19 binding Fab molecule.

For correct pairing of the heavy chains (formation of a heterodimericmolecule), knob-into-hole mutations were introduced in the constantregion of the antibody heavy chains (T366W/S354C andT366S/L368A/Y407V/Y349C, respectively).

Furthermore, the P329G, L234A and L235A mutations were introduced in theconstant region of the antibody heavy chains to abrogate binding to Fcγreceptors.

The TCBs were prepared by Evitria (Switzerland) using their proprietaryvector system with conventional (non-PCR based) cloning techniques andusing suspension-adapted CHO K1 cells (originally received from ATCC andadapted to serum-free growth in suspension culture at Evitria). For theproduction, Evitria used its proprietary, animal-component free andserum-free media (eviGrow and eviMake2) and its proprietary transfectionreagent (eviFect). The cells were transfected with the correspondingexpression vectors in a 1:1:2:1 (“vector knob heavy chain”:“vector holeheavy chain”:“vector CD3 light chain”:“vector CD19 light chain”).Supernatant was harvested by centrifugation and subsequent filtration(0.2 μm filter).

As an alternative to the preparation at Evitria, TCB molecules wereprepared in-house by transient transfection of HEK293 EBNA cells. Cellswere centrifuged and, medium was replaced by pre-warmed CD CHO medium(Thermo Fisher, #10743029). Expression vectors were mixed in CD CHOmedium, polyethylenimine (PEI; Polysciences Inc, #23966-1) was added,the solution vortexed and incubated for 10 minutes at room temperature.Afterwards, cells (2 mio/ml) were mixed with the vector/PEI solution,transferred to a flask and incubated for 3 hours at 37° C. in a shakingincubator with a 5% CO₂ atmosphere. After the incubation, Excell mediumwith supplements (80% of total volume) was added (W. Zhou and A.Kantardjieff, Mammalian Cell Cultures for Biologics Manufacturing, DOI:10.1007/978-3-642-54050-9; 2014). One day after transfection,supplements (Feed, 12% of total volume) were added. Cell supernatantswere harvested after 7 days by centrifugation and subsequent filtration(0.2 μm filter).

Proteins were purified from the harvested supernatant by standardmethods. In brief, Fc containing proteins were purified from filteredcell culture supernatants by Protein A-affinity chromatography(equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was achievedat pH 3.0 followed by immediate pH neutralization of the sample. Theprotein was concentrated by centrifugation (Millipore Amicon® ULTRA-15,#UFC903096), and aggregated protein was separated from monomeric proteinby size exclusion chromatography in 20 mM histidine, 140 mM sodiumchloride, pH 6.0.

The concentrations of purified proteins were determined by measuring theabsorption at 280 nm using the mass extinction coefficient calculated onthe basis of the amino acid sequence according to Pace, et al., ProteinScience, 1995, 4, 2411-1423. Purity and molecular weight of the proteinswere analyzed by CE-SDS in the presence and absence of a reducing agentusing a LabChipGXII (Perkin Elmer). Determination of the aggregatecontent was performed by HPLC chromatography at 25° C. using analyticalsize-exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated inrunning buffer (25 mM K₂HPO₄, 125 mM NaCl, 200 mM L-argininemonohydrocloride, pH 6.7 or 200 mM KH₂PO₄, 250 mM KCl pH 6.2,respectively).

Results from the biochemical and biophysical analysis of the preparedTCB molecules are given in Table 1.

All four TCB molecules could be produced in good quality.

TABLE 1 Biochemical and biophysical analysis of anti-CD3 antibodyCD3_(opt) in CD19-TCB format. Monomer content determined by analyticalsize exclusion chromatography. Purity determined by non-reducing CE-SDS.Molecule Monomer [%] Purity [%] CD19(018) CD3_(orig) 91 97 CD19(2B11)CD3_(orig) 96 97 CD19(2B11) CD3_(opt) 98 99 CD19(018) CD3_(opt) 98 99

Example 5—Functional Characterization of Optimized Anti-CD3(Multispecific) Antibodies by Surface Plasmon Resonance (SPR)

SPR experiments were performed, using the CD19-TCB molecules prepared inExample 4, on a Biacore T200 instrument at 25° C. with HBS-EP+ asrunning buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% Surfactant P20(GE Healthcare, #BR-1006-69)). Anti-Fc(P329G) IgG (an antibody thatspecifically binds human IgG₁ Fc(P329G); “anti-PG antibody”—see WO2017/072210, incorporated herein by reference) was directly immobilizedby amine coupling on a C1 chip (GE Healthcare). The different TCBmolecules were captured at 25 nM. Three-fold dilution series (in HBS-EPfrom 0.14 to 100 nM) of the CD19 antigen (human CD19 extracellulardomain (ECD)—Fc fusion; see SEQ ID NOs 43 and 44) or the CD3 antigen(CD3ε/δ—Fc fusion; see Example 2, SEQ ID NOs 41 and 42) were passed overthe ligand at 30 μl/min for 240 s to record the association phase. Thedissociation phase was monitored for 1500 s (CD19 antigen) or 800 s (CD3antigen)s and triggered by switching from the sample solution toHBS-EP+. The chip surface was regenerated after every cycle using twoinjections of 10 mM glycine pH 2.1 for 60 sec. Bulk refractive indexdifferences were corrected for by subtracting the response obtained onthe reference flow cell (without captured TCB). The affinity constantswere derived from the kinetic rate constants by fitting to a 1:1Langmuir binding using the Biaeval software (GE Healthcare). Themeasurement was performed with three independent dilution series.

The kinetic constants for a 1:1 Langmuir binding were determined for thefour tested TCBs to recombinant human CD19 (Table 2) and to recombinanthuman CD3 (Table 3).

TABLE 2 Binding to human CD19: Kinetic constants. Average and standarddeviation (in parenthesis) of independent dilutions series in the samerun. t_(1/2) Molecule k_(on) (1/Ms) k_(off) (1/s) K_(D) (M) (min)CD19(018) CD3_(orig) 3.33E+05 3.16E−04 9.5E−10  36.6 (1.17E+04)(9.54E−06) (6E−11) CD19(2B11) CD3_(orig) 4.34E+05 1.76E−04 4.0E−10  65.6(1.20E+04) (5.77E−07) (1E−11) CD19(2B11) CD3_(opt) 4.10E+05 1.46E−043.6E−10  79.1 (1.10E+04) (1.53E−06) (1E−11) CD19(018) CD3_(opt) 3.35E+053.83E−04 1.14E−09   30.2 (9.64E+03) (7.37E−06) (6E−11)

TABLE 3 Binding to human CD3: Kinetic constants. Average and standarddeviation (in parenthesis) of independent dilutions series in the samerun. t_(1/2) Molecule k_(on) (1/Ms) k_(off) (1/s) K_(D) (M) (min)CD19(018) CD3_(orig) 9.33E+05 3.33E−03 3.57E−09  3.5 (2.57E+04)(1.01E−04) (1.7E−10) CD19(2B11) CD3_(orig) 1.13E+06 3.78E−03 3.36E−09 3.1 (5.77E+03) (1.10E−04) (1.1E−10) CD19(2B11) CD3_(opt) 2.37E+068.38E−03 3.55E−09  1.4 (1.93E+05) (1.49E−04) (2.2E−10) CD19(018)CD3_(opt) 4.39E+06 1.30E−02 2.98E−09  0.9 (7.22E+05) (1.00E−03)(2.5E−10)

The different binders show similar affinities in the different TCBs. TheCD19 binder 2B11 has a K_(D) of around 0.4 nM in the respective TCBs.The CD19 binder 018 is of slightly lower affinity with a K_(D) around1.1 nM in the respective TCBs. The TCBs with CD3 binder CD3_(orig) orCD3_(opt) have comparable affinity to CD3 with a K_(D) around 3.4 nM.While the K_(D) of the interaction with CD19 and CD3 antigens aresimilar, the kinetic is different. CD19 dissociates slower than CD3does, but CD3 associates faster than CD19 does, thus leading to similarK_(D) values.

Example 6—Binding of CD19-TCB Molecules with Optimized Anti-CD3 Antibodyto Human CD19- and Human CD3-Expressing Cells

The binding of the CD19-TCB molecules prepared in Example 4 to humanCD19- and CD3-expressing target cells was tested. Two CD19-expressingcell lines with different levels of CD19 expression were used. Nalm-6,an acute lymphoblastic leukemia (ALL) cell line with high CD 19expression and Z-138 (Mantle cell lymphoma) with average expressionlevels. CD3-binding was assessed using immortalized T lymphocyte line(Jurkat cell line). Briefly, cells were harvested, counted, checked forviability and resuspended at 1×10⁶ cells/ml in FACS buffer (PBS+2% FCS+5mM EDTA+0.25% sodium azide). 100 μl of cell suspension (containing0.1×10⁶ cells) were incubated in round-bottom 96-well plate for 30 minat 4° C. with increasing concentrations of CD19-TCB molecules (200nM-0.05 nM on Jurkat cells; 200 nM-0.0002 nM on Z-128 and Nalm-6 cells),washed twice with cold FACS buffer, re-incubated for further 30 min at4° C. with the PE-conjugated AffiniPure F(ab′)2 Fragment goat anti-humanIgG Fcγ fragment specific secondary antibody (Jackson Immuno ResearchLab PE #109-116-170), washed twice with cold FACS buffer and immediatelyanalyzed by FACS using a FACS CantoII (software FlowJo 10.5.3). Bindingcurves and the EC50 values related to binding were calculated usingGraphPad Prism 7.

The results are shown in FIG. 7 and Table 4 and 5. The CD19-TCBmolecules comprising CD3_(opt) as CD3 binder show comparable (orslightly better) CD3 binding to molecules comprising the CD3 binderCD3_(orig) (FIG. 7A and Table 4).

The CD19-TCB molecules (comprising either 2B11 or 018 as CD19 binders)show comparable binding to CD19-expressing cells (FIG. 7B, C and Table5). For Z-138 cells, the EC50 values could not be calculated since thebinding curve did not reach saturation.

TABLE 4 EC50 values (nM) of binding of CD19-TCBs to human CD3-expressingJurkat cells. Antibodies EC50 [nM] CD19(2B11) CD3_(opt) 53.45 CD19(018)CD3_(opt) 126.5 CD19(2B11) CD3_(orig) 203.4 CD19(018) CD3_(orig) 115.1

TABLE 5 EC50 values (nM) of binding of CD19-TCBs to humanCD19-expressing target Nalm-6 cells. Antibodies EC50 [nM] CD19(2B11)CD3_(opt) 0.6 CD19(018) CD3_(opt) 1 CD19(2B11) CD3_(orig) 0.7 CD19(018)CD3_(orig) 1.5

Example 7—Tumor Cell Lysis and T Cell Activation, Induced by CD19-TCBMolecules with Optimized Anti-CD3 Antibody

The lysis of CD19-expressing tumor cells and subsequent T cellactivation mediated by the CD19-TCB molecules prepared in Example 4 wasassessed on Nalm-6 cells (ALL) and Z-138 cells (Mantle cell lymphoma).Human PBMCs were used as effectors and tumor lysis was detected at 20 hof incubation with the different TCB molecules.

Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaquedensity centrifugation of enriched lymphocyte preparations (buffy coats)obtained from a healthy human donor. Fresh blood was diluted withsterile PBS and layered over Histopaque gradient (Sigma, #H8889). Aftercentrifugation (450×g, 30 minutes, no brake, room temperature), theplasma above the PBMC-containing interphase was discarded and PBMCstransferred in a new falcon tube subsequently filled with 50 ml of PBS.The mixture was centrifuged (350×g, 10 minutes, room temperature), thesupernatant discarded and the PBMC pellet incubated in erythrocytelysing solution for 5 min at 37° C. before washing with sterile PBS(centrifugation 300×g, 10 minutes). The resulting PBMC population wasresuspended in PBS and counted automatically (ViCell). 50 mio PBMCs percyrovial were frozen in RPMI1640 medium (Gibco, #21870076) containing10% FCS and 1% GlutaMAX (Gibco) containing 10% DMSO (Sigma, #D2650).PBMCs were thawed the day of the assay and counted again automatically(ViCell). The amount needed was washed once with sterile PBS. B celldepletion was performed using CD20 microbeads (Miltenyi, #130-091-104)according to the manufacturer's instructions. B cell depleted PBMCs werecounted (ViCell) and resuspended at 5×10⁶ cells/ml in RPMI1640 mediumcontaining 10% FCS and 1% GlutaMAX.

For the killing assay, 0.25 mio B cell-depleted PBMCs were added to theU-bottom 96-well plates. Briefly, target cells were harvested, washed,and plated at density of 50 000 cells/well resulting in an finaleffector-to-target (E:T) ratio of 5:1. The TCB molecules were added atthe indicated concentrations (range of 0.02 pM-1000 pM, in triplicates).CD107a (LAMP-1) was directly stained already in the assay (PE anti-humanCD107a; Biolegend, #328608).

Tumor cell lysis was assessed after 20 h of incubation at 37° C., 5% CO₂by quantification of LDH released into cell supernatants byapoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11644 793 001). Maximal lysis of the target cells (=100%) was achieved byincubation of target cells with 1% Triton X-100. Minimal lysis (=0%)refers to target cells co-incubated with effector cells withoutbispecific construct.

For the assessment of T cell activation occurring upon tumor cell lysis,PBMCs were centrifuged at 400×g for 4 min and washed twice with FACSbuffer. Briefly, cells were washed twice with PBS, followed by live/deadstaining (Zombie Aqua Fixable Viability kit; Biolegend, #423102, 20 minat RT). After repeated washing first with PBS followed by FACS Bufferthe surface staining for CD3 (PE-Cy5 anti-human CD3; BD Pharmigen,#555341), CD4 (BV605 anti-human CD4; Biolegend, #317438), CD8 (BV711anti-human CD8; Biolegend, #301044), CD25 (PE-Cy7 anti-human CD25;Biolegend, #302612) and CD69 (BV421 anti-human CD69; Biolegend, #310930)was performed according to the suppliers' indications. Cells were washedtwice with 150 μl/well FACS buffer and fixed with 120 μl/well 1× lysingsolution (BD Biosciences #349202). Samples were analyzed at BD FACSFortessa (Software FlowJo 10.5.3).

FIG. 8 shows that CD19-TCB molecules induced target-specific killing ofCD19+ target cells. The four different CD19-TCB molecules were overallcomparable in inducing lysis of CD19-expressing tumor cells. FIGS. 9 and10 show that the CD19-TCBs containing the CD3_(opt) binder show slightlysuperior induction of T cell activation after tumor killing (CD25, CD69and CD107 expression on CD8 and CD4 T cells upon killing of Nalm-6 orZ-138 target cells) compared to molecules containing the CD3_(orig)binder. No effect on T cell activation of the CD19 binders 2B11 and 018was observed.

TABLE 6 EC50 values (pM) of tumor cell lysis mediated by CD19-TCBmolecules evaluated on CD19-expressing tumor target cells. EC50 (pM)Nalm-6 Z-138 CD19(2B11) CD3_(opt) 0.24 2.9 CD19(018) CD3_(opt) 0.4 3CD19(2B11) CD3_(orig) 0.3 4.8 CD19(018) CD3_(orig) 0.3 5.4

TABLE 7 EC50 values (pM) of T cell activation upon tumor cell lysismediated by CD19-TCB molecules using Z-138 as target cells. ActivationCD19(2B11) CD19(018) CD19(2B11) CD19(018) marker CD3_(opt) CD3_(opt)CD3_(orig) CD3_(orig) CD25/CD4 1.3 1.4 2 2 CD69/CD4 0.3 0.5 0.6 0.7CD107a/CD4 0.6 0.8 1.3 1.4 CD25/CD8 0.9 1 1.5 1.7 CD69/CD8 0.2 0.4 0.60.8 CD107a/CD8 0.5 0.7 1.3 1.4

TABLE 8 EC50 values (pM) of T cell activation upon tumor cell lysismediated by CD19-TCB molecules using Nalm-6 as target cells. ActivationCD19(2B11) CD19(018) CD19(2B11) CD19(018) marker CD3_(opt) CD3_(opt)CD3_(orig) CD3_(orig) CD25/CD4 8 11 12 7 CD69/CD4 0.9 1.2 1.9 1.7CD107a/CD4 4.1 3.6 6.9 5.5 CD25/CD8 2.1 2.4 3.4 2.8 CD69/CD8 0.2 0.4 0.70.4 CD107a/CD8 1.2 1.4 2.1 1.9

Example 8—Determination of Thermal Stability of CD19-TCB Molecule withOptimized Anti-CD3 Antibody

Thermal stability of the CD19-TCB molecule comprising the optimizedanti-CD3 antibody CD3_(opt) (and the CD19 binder 2B11, see Example 4)was monitored by Static Light Scattering (SLS) by applying a temperatureramp using an Uncle system (Unchained Labs, USA).

9 μl of filtered protein sample with a protein concentration of 1 mg/mlwas applied to the Uncle device. The temperature was ramped from 30 to90° C. at 0.1° C./min, with scattering intensity at 266 nm beingcollected.

The result is shown in Table 9.

TABLE 9 Thermal stability of CD19-TCB molecule as measured by staticlight scattering. anti-CD3 antibody T_(agg) [° C.] CD3_(opt) 64.8

Example 9—Characterization of CD19-TCB Molecule with Optimized Anti-CD3Antibody by Surface Plasmon Resonance (SPR) after Stress

In order to confirm the effect of the deamidation site removal and itseffect on the stability of the antibody, the CD19-TCB moleculecomprising the optimized anti-CD3 antibody CD3_(opt) (and the CD19binder 2B11, see Example 4) was incubated for 14 days at 37° C., pH 7.4and at 40° C., pH 6 and further analyzed by SPR for their bindingcapability to human CD3ε/δ. Samples stored at −80° C. pH 6 were used asreference. The reference samples and the samples stressed at 40° C. werein 20 mM His, 140 mM NaCl, pH 6.0, and the samples stressed at 37° C. inPBS, pH 7.4, all at a concentration of 1.0 mg/ml. After the stressperiod (14 days) samples in PBS were dialyzed back to 20 mM His, 140 mMNaCl, pH 6.0 for further analysis.

All SPR experiments were performed on a Biacore T200 instrument (GEHealthcare) at 25° C. with HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4,0.05% Surfactant P20) as running and dilution buffer. Biotinylated humanCD3ε/δ (see Examples 2, SEQ ID NOs 41 and 42) as well as biotinylatedanti-huIgG (Capture Select, Thermo Scientific, #7103262100) wereimmobilized on a Series S Sensor Chip SA (GE Healthcare, #29104992),resulting in surface densities of at least 1000 resonance units (RU).TCBs with a concentration of 2 μg/ml were injected for 30 s at a flowrate of 5 μl/min, and dissociation was monitored for 120 s. The surfacewas regenerated by injecting 10 mM glycine pH 1.5 for 60 s. Bulkrefractive index differences were corrected by subtracting blankinjections and by subtracting the response obtained from a blank controlflow cell. For evaluation, the binding response 5 seconds afterinjection end was taken. To normalize the binding signal, the CD3binding was divided by the anti-huIgG response (the signal (RU) obtainedupon capture of the TCB on the immobilized anti-huIgG antibody). Therelative binding activity was calculated by referencing each temperaturestressed sample to the corresponding, non-stressed sample.

As shown in Table 10, binding of the CD19-TCB comprising the optimizedanti-CD3 binder CD3_(opt) to CD3ε/δ is essentially unaffected uponstress, in line with the results for the CD3 binder as such (Example 2).

TABLE 10 Binding activity of CD19-TCB molecule to human CD3ε/δ afterincubation at pH 6/40° C. or pH 7.4/37° C. for 2 weeks. binding activity[%] 2 weeks at 2 weeks at anti-CD3 antibody pH 6.0/40° C. pH 7.4/37° C.CD3_(opt) 98 98

Example 10—In Vivo B Cell Depletion and Cytokine Release Induced byCD19-TCB Molecule with Optimized Anti-CD3 Antibody

To understand the potency and safety profile of the CD19-TCB moleculecomprising the the optimized anti-CD3 antibody CD3_(opt) and the 2B11CD19 binder, an in vivo mode of action study was conducted assessingperipheral B cell depletion and cytokine release in humanized NSG mice.

Female NSG mice, age 4-5 weeks at start of the experiment (JacksonLaboratory), were maintained under specific-pathogen-free condition withdaily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). The experimental study protocolwas reviewed and approved by local government (ZH223-17). After arrival,animals were maintained for one week to get accustomed to the newenvironment and for observation. Continuous health monitoring wascarried out on a regular basis.

Female NSG mice were injected i.p. with 15 mg/kg of Busulfan followedone day later by an i.v. injection of 1×10⁵ human hematopoietic stemcells isolated from cord blood. At week 14-16 after stem cell injection,mice were bled sublingual and blood was analyzed by flow cytometry forsuccessful humanization. Efficiently engrafted mice were randomizedaccording to their human T cell frequencies into the different treatmentgroups. Following randomization, mice from three groups were pre-treatedonce with obinutuzumab (Gazyva®) (30 mg/kg), as a measure to preventexcessive cytokine release.

7 days after this pre-treatment, on day 0, all groups received theCD19-TCB at different doses, CD20-TCB (a TCB targeting CD20 andcomprising the CD3 binder CD3_(orig)), or vehicle. Three different doses(0.5, 0.15 and 0.05 mg/kg) of CD19-TCB were injected. CD20-TCB (0.15mg/kg) with and without Gazyva® pre-treatment was used as comparisonagent. All mice were injected i.v. with 200 μl of the appropriatesolution. Three mice per group were bled at 4 h, 24 h and 72 h aftertherapy (day 0).

The study design is shown in FIG. 11, and the study groups aresummarized in Table 11.

TABLE 11 Study groups (number of animals per group = 3, treatmentadministration = i.v.) Group Treatment Dose (mg/kg) A Vehicle — BCD19-TCB 0.5 C CD19-TCB 0.15 D CD19-TCB 0.05 E Obinutuzumab (Gazyva ®)30 CD19-TCB 0.5 F CD20-TCB 0.15 G Obinutuzumab (Gazyva ®) 30 CD20-TCB0.15

At termination (day 3), mice were sacrificed and spleen, lymph nodes(LN) and bone marrow (BM) were harvested, weighed, and single cellsuspensions were prepared through an enzymatic digestion with Liberaseand DNAse for subsequent FACS analysis. Spleen single cells as well asall blood samples were stained for human CD45, CD19, CD20 and analyzedat the BD Fortessa flow cytometer. Additionally, serum from the threebleeding time points were analyzed for cytokine content by Multiplexanalysis.

FIG. 12 shows the body weight change (%) in the treatment groups. TheCD19-TCB molecule induced less body weight drop compared to CD20-TCBtreatment. This body weight drop was independent of the dose used.Furthermore, cytokine analysis in the sera of treated animals revealed apeak of elevated cytokine levels at 4 h after treatment with theCD20-TCB molecule (which could be reduced by pre-treatment with Gazyva®(GPT)), whereas only low levels of cytokines were detected for theCD19-TCB molecule (FIG. 13).

The Immuno-PD data (FIG. 14) on the kinetics of B cell depletion inblood revealed strong depletion of CD19+CD20+B cells (mean counts ofCD19+ or CD20+ cells/μl blood+/−SEM) over time by CD19-TCB and suggesteda dose-dependency. This B cell depletion effect was also seen in alllymphatic organs analyzed at 72 h upon treatment with CD19-TCB being aspotent as CD20-TCB (data not shown).

Example 11—Tumor Growth Control in Mouse Xenograft Experiment byCD19-TCB Molecule with Optimized Anti-CD3 Antibody

To evaluate the anti-tumor efficacy of CD19-TCB molecule comprising thethe optimized anti-CD3 antibody CD3_(opt) and the 2B11 CD19 binder invivo, humanized NSG mice were engrafted with CD19+ lymphoma patientderived xenograft (PDX) cells from a patient who relapsed R-CHOPtreatment. When the tumor volume reached 200 mm³, mice were randomizedin groups of 8 based on their tumor size. They were then weekly injectedwith 0.5 mg/kg CD19-TCB or vehicle (i.v.) as illustrated in FIG. 15. Toassess the effect of CD19-TCB on tumor growth, tumor volumes werecalculated from caliper measurements twice or three times per week.

As a result, weekly treatment CD19-TCB exerted a significant tumorgrowth control as compared to treatment with vehicle (FIG. 16). Thisdata demonstrate that the weekly dosing with 0.5 mg/kg CD19-TCB isefficacious in lymphoma PDX-bearing huNSG mice, and suggest thatlymphoma patients relapsing R-CHOP treatment could benefit from CD19-TCBtreatment.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. An antibody that binds to CD3 and CD19, wherein the antibodycomprises (a) a first antigen binding domain that binds to CD3,comprising a heavy chain variable region (VH) comprising a heavy chaincomplementary determining region (HCDR) 1 of SEQ ID NO: 2, a HCDR 2 ofSEQ ID NO: 3, and a HCDR 3 of SEQ ID NO: 5, and a light chain variableregion (VL) comprising a light chain complementarity determining region(LCDR) 1 of SEQ ID NO: 8, a LCDR 2 of SEQ ID NO: 9 and a LCDR 3 of SEQID NO: 10; and (b) a second antigen binding domain that binds to CD19.2. The antibody of claim 1, wherein the VH of the first antigen bindingdomain comprises an amino acid sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and/or the VL of the first antigen binding domain comprises an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO:
 11. 3. An antibodythat binds to CD3 and CD19, wherein the antibody comprises (a) a firstantigen binding domain that binds to CD3 comprising a VH sequence of SEQID NO: 7 and a VL sequence of SEQ ID NO: 11; and (b) a second antigenbinding domain that binds to CD19.
 4. The antibody of claim 1, whereinthe first and/or the second antigen binding domain is a Fab molecule. 5.The antibody of claim 1, comprising an Fc domain composed of a first anda second subunit.
 6. The antibody of claim 1, wherein the first antigenbinding domain is a Fab molecule wherein the variable domains VL and VHor the constant domains CL and CH1, particularly the variable domains VLand VH, of the Fab light chain and the Fab heavy chain are replaced byeach other.
 7. The antibody of claim 1, wherein the second antigenbinding domain is a conventional Fab molecule.
 8. The antibody of claim1, wherein the second antigen binding domain is a Fab molecule whereinin the constant domain CL the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 the amino acid atposition 147 is substituted independently by glutamic acid (E), oraspartic acid (D) (numbering according to Kabat EU index) and the aminoacid at position 213 is substituted independently by glutamic acid (E),or aspartic acid (D) (numbering according to Kabat EU index).
 9. Theantibody of claim 1, wherein the first and the second antigen bindingdomain are fused to each other, optionally via a peptide linker.
 10. Theantibody of claim 1, wherein the first and the second antigen bindingdomain are each a Fab molecule and either (i) the second antigen bindingdomain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding domain,or (ii) the first antigen binding domain is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding domain.
 11. The antibody of claim 1, wherein thefirst and the second antigen binding domain are each a Fab molecule andthe antibody comprises an Fc domain composed of a first and a secondsubunit; and wherein either (i) the second antigen binding domain isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first antigen binding domain and the firstantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first subunit of the Fc domain, or (ii) thefirst antigen binding domain is fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the second antigenbinding domain and the second antigen binding domain is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first subunitof the Fc domain.
 12. The antibody of claim 5, wherein the Fc domain isan IgG, particularly an IgG₁, Fc domain.
 13. The antibody of claim 5,wherein the Fc domain is a human Fc domain.
 14. The antibody of claim 5,wherein the Fc domain comprises a modification promoting the associationof the first and the second subunit of the Fc domain.
 15. The antibodyof claim 5, wherein the Fc domain comprises one or more amino acidsubstitution that reduces binding to an Fc receptor and/or effectorfunction.
 16. The antibody of claim 1, wherein the second antigenbinding domain comprises (i) a VH comprising a HCDR 1 of SEQ ID NO: 15,a HCDR 2 of SEQ ID NO: 16, and a HCDR 3 of SEQ ID NO: 17, and a VLcomprising a LCDR 1 of SEQ ID NO: 19, a LCDR 2 of SEQ ID NO: 20 and aLCDR 3 of SEQ ID NO: 21; or (ii) a VH comprising a HCDR 1 of SEQ ID NO:28, a HCDR 2 of SEQ ID NO: 29, and a HCDR 3 of SEQ ID NO: 30, and a VLcomprising a LCDR 1 of SEQ ID NO: 32, a LCDR 2 of SEQ ID NO: 33 and aLCDR 3 of SEQ ID NO:
 34. 17. The antibody of claim 1, wherein the secondantigen binding domain comprises (i) a VH comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 18, and/or a VLcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22;or (ii) a VH comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 31, and/or a VL comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:
 35. 18. An isolated polynucleotide encoding theantibody of claim
 1. 19. A host cell comprising the isolatedpolynucleotide of claim
 18. 20. A method of producing an antibody thatbinds to CD3 and CD19, comprising the steps of (a) culturing the hostcell of claim 19 under conditions suitable for the expression of theantibody and optionally (b) recovering the antibody.
 21. An antibodythat binds to CD3 and CD19 produced by the method of claim
 20. 22. Apharmaceutical composition comprising the antibody of claim 1 or 21 anda pharmaceutically acceptable carrier.
 23. (canceled)
 24. A method oftreating a disease comprising the antibody of claim 1 or
 21. 25. Themethod of claim 24, wherein the disease is cancer or an autoimmunedisease. 26-28. (canceled)
 29. A method of treating a disease in anindividual, comprising administering to said individual an effectiveamount of the antibody of claim 1 or
 21. 30. The method of claim 29,wherein the disease is cancer or an autoimmune disease.
 31. (canceled)32. The antibody claim 1, wherein the antibody further comprises a thirdantigen binding domain that binds to CD19.
 33. The antibody of claim 3,wherein the antibody further comprises a third antigen binding domainthat binds to CD19.
 34. The antibody of claim 32, wherein the first, thesecond and/or the third antigen binding domain is a Fab molecule. 35.The antibody of claim 34, wherein the second antigen binding domain is aconventional Fab molecule and the third antigen binding domain is aconventional Fab molecule.
 36. The antibody of claim 32, wherein each ofthe second antigen binding domain and the third antigen binding domainis a Fab molecule wherein in the constant domain CL the amino acid atposition 124 is substituted independently by lysine (K), arginine (R) orhistidine (H) (numbering according to Kabat) and the amino acid atposition 123 is substituted independently by lysine (K), arginine (R) orhistidine (H) (numbering according to Kabat), and in the constant domainCH1 the amino acid at position 147 is substituted independently byglutamic acid (E), or aspartic acid (D) (numbering according to Kabat EUindex) and the amino acid at position 213 is substituted independentlyby glutamic acid (E), or aspartic acid (D) (numbering according to KabatEU index).
 37. The antibody of claim 32, wherein the first antigenbinding domain is a Fab molecule, the second antigen binding domain is aFab molecule, and the third antigen binding domain is a Fab molecule andthe antibody comprises an Fc domain composed of a first and a secondsubunit; and wherein either (i) the second antigen binding domain isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first antigen binding domain and the firstantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first subunit of the Fc domain, or (ii) thefirst antigen binding domain is fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the second antigenbinding domain and the second antigen binding domain is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first subunitof the Fc domain; and the third antigen binding domain, where present,is fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe second subunit of the Fc domain.
 38. The antibody of claim 32,wherein the second antigen binding domain and the third antigen bindingdomain each comprises (i) a VH comprising a HCDR 1 of SEQ ID NO: 15, aHCDR 2 of SEQ ID NO: 16, and a HCDR 3 of SEQ ID NO: 17, and a VLcomprising a LCDR 1 of SEQ ID NO: 19, a LCDR 2 of SEQ ID NO: 20 and aLCDR 3 of SEQ ID NO: 21; or (ii) a VH comprising a HCDR 1 of SEQ ID NO:28, a HCDR 2 of SEQ ID NO: 29, and a HCDR 3 of SEQ ID NO: 30, and a VLcomprising a LCDR 1 of SEQ ID NO: 32, a LCDR 2 of SEQ ID NO: 33 and aLCDR 3 of SEQ ID NO:
 34. 39. The antibody of claim 32, wherein thesecond antigen binding domain and the third antigen binding domain eachcomprises (i) a VH comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 18, and/or a VL comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 22; or (ii) a VH comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 31, and/or a VLcomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35.40. An isolated polynucleotide encoding the antibody of claim
 32. 41. Ahost cell comprising the isolated polynucleotide of claim
 40. 42. Amethod of producing an antibody that binds to CD3 and CD19, comprisingthe steps of (a) culturing the host cell of claim 41 under conditionssuitable for the expression of the antibody and optionally (b)recovering the antibody.
 43. An antibody that binds to CD3 and CD19produced by the method of claim
 42. 44. A pharmaceutical compositioncomprising the antibody of claim 32 or claim 43 and a pharmaceuticallyacceptable carrier.
 45. A method of treating a disease comprising theantibody of claim 32 or claim
 43. 46. The method of claim 45, whereinthe disease is cancer or an autoimmune disease.
 47. A method of treatinga disease comprising the pharmaceutical composition of claim
 44. 48. Themethod of claim 47, wherein the disease is cancer or an autoimmunedisease.
 49. A method of treating a disease in an individual, comprisingadministering to said individual an effective amount of the antibody ofclaim 32 or claim
 43. 50. The method of claim 49, wherein the disease iscancer or an autoimmune disease.
 51. A method of treating a disease inan individual, comprising administering to said individual an effectiveamount of the pharmaceutical composition of claim
 44. 52. The method ofclaim 51, wherein the disease is cancer or an autoimmune disease.
 53. Amethod of treating a disease comprising the pharmaceutical compositionof claim
 22. 54. The method of claim 53, wherein the disease is canceror an autoimmune disease.
 55. A method of treating a disease in anindividual, comprising administering to said individual an effectiveamount of the pharmaceutical composition of claim
 22. 56. The method ofclaim 55, wherein the disease is cancer or an autoimmune disease.